Search

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

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

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

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

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

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. The origin, evolution and domestication of Citrus and the genealogy of the most important wild and cultivated citrus varieties. Citrus fruits are one of the most cultivated crops worldwide, yet the evolutionary relationships among citrus species remain uncertain. Daniel Rokhsar, Manuel Talon and colleagues analyse the genomes of 60 accessions that represent a diverse range of citrus species, including 30 newly sequenced citrus genomes. They characterize the diversity and evolution of citrus at the species level and identify interspecific citrus hybrids and admixtures—genetic mixing between previously isolated populations—that could be the result of human activities such as migration and agriculture. The authors identify 10 progenitor species and suggest that citrus originated in southeast Asia, diversifying during the late Miocene epoch through a rapid southeast Asian radiation that correlated with a changing climate, including the weakening of the monsoons. They also find extensive relatedness among mandarins and sweet oranges, showing a complex history of admixture during the domestication of these groups.

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

Quality in citrus is mainly characterized by fruit and juice colour, fruit and skin size, juice percent, total soluble solids, titrable acidity, and carotenoid/flavonoid contents. Moreover, studies of biosynthetic pathway of the metabolites/proteins involved in quality at transcriptional and translational levels may give relevant information for subsequent functional studies and quality improvement. Data mining of ESTs from HarvEST database allowed the selection of 17 cDNA libraries from albedo, flavedo, peel, pulp and juice sac of different orange, mandarin, clementine and grapefruit varieties. In order to select key genes involved in quality we used systems biology that offers mathematical tools that include the analysis of the structure, clustering and centralities of the network. In order to have information regarding physical protein-protein interactions (PPPI) from citrus sequences, orthologous sequences of Arabidopsis thaliana were used (BLASTX; reciprocal BLASTP). Literature data mining was performed, and PPPI network design was obtained using the Cytoscape software. The interactome networks thus obtained were analyzed with MCODE. Gene ontology clustering analysis was performed using BiNGO. Specific algorithms were applied to identify modules and central nodes within the citrus libraries associated network. The obtained results will be used as a guideline to select specific genes/proteins from citrus for further functional studies as gene expression or plant transformation. (Résumé)

In citrus, the use of rootstocks promotes productivity, improves fruit quality and may confer resistance or tolerance to biotic and abiotic stress. 'Rangpur' lime (Citrus limonia, Osbeck) is one of the most tolerant rootstock to drought and is largely used by the Brazilian citrus industry. Previous studies have shown that, when grafted with a variety citrus doubled diploid (4x) 'Rangpur' lime rootstock is more tolerant to drought stress than the respective diploid (2x). In the present study, we characterized the water deficit tolerance in 2x and 4x 'Rangpur' lime seedlings. Water deficit was applied respectively for 21 days, followed by recovery irrigation. Physiological and biochemical parameters were measured periodically during the experiment and samples were collected for biochemical analysis. Doubled diploid seedlings were showed to be more water deficit tolerant than 2x. Water deficit caused a greater reduction in stomatal conductance in 2x compared to 4x. The 21st day of stress, leaf water potential in 2x was lower. Analyses of the activity of enzymes involved in detoxification process suggest that the better tolerance of 4x genotypes may be related to better scavenging of reactive oxygen species. (Resume d'auteur)

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

Cybridization should be a way to induce phenotypical variations in highly heterozygous secondary citrus species or genotypes, without breaking their very complex multilocus structure by sexual recombination. It should have particular interest to manipulate the nucleo-cytoplasmic male sterility, to transfer tolerance traits with cytoplasmic determinisms and eventually to improve tolerance to oxidative stress. The present work is focused on the creation of new cybrids through three different approaches of protoplast fusion: (i) symmetric hybridization, (ii) donor-recipient protoplast fusion by UV irradiation of donor protoplasts, and (iii) cytoplasts + protoplast fusion. Sixteen combinations of symmetric protoplast fusion have produced cybrids. No cybrid was obtained after UV irradiation of donor protoplasts while three combinations of cytoplasts + protoplast fusion produced cybrids. Therefore, cytoplast fusion appears much more promising than the UV approach for targeted cybridization. The innovative plant material is currently propagated for further cultivar and rootstock selection and for basic researches on the impact of nucleo-cytoplasmic interaction on desirable traits. (Resume d'auteur)

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

Breeding and selection of new citrus rootstocks are nowadays of the utmost importance in the Mediterranean Basin because the citrus industry faces increasing biotic and abiotic constraints. In Tunisia, citrus contributes significantly to the national economy, and its extension is favored by natural conditions and economic considerations. Sour orange, the most widespread traditional rootstock of the Mediterranean area, is also the main one in Tunisia. In addition to sour orange, other citrus rootstocks well adapted to local environmental conditions are traditionally used and should be important genetic resources for breeding. Prior to initiation of any breeding program, the exploration of Tunisian citrus rootstock diversity was a priority. Two hundred and one local accessions belonging to four facultative apomictic species (Citrus aurantium, sour orange; Citrus sinensis, sweet orange; Citrus limon, lemon; and Citrus aurantifolia, lime) were collected and genotyped using 20 nuclear SSR markers and four InDel mitochondrial markers. Sixteen distinct Multi-locus genotypes (MLGs) were identified and compared to references from French and Spanish collections. The differentiation of the four varietal groups was well-marked. Each group displayed a relatively high allelic diversity, primarily due to very high heterozygosity. 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. This diversity study enabled the identification of a core sample of accessions for further physiological and agronomic evaluations. These core accessions will be integrated into citrus rootstock breeding programs for the Mediterranean Basin. (Resume d'auteur)

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

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

Citrus triploid hybrids can be recovered by 2x X 2x hybridisations as a consequence of the formation of 2n gametes or by interploid hybridisation. Most of the 4x parents used in our program are doubled-diploid (DD), also known as autotetraploid. The genetic structure of diploid gametes and particularly the rate of parental heterozygosity restitution (PHR) depend on the meiotic process by which they were originated. Second-division restitution (SDR) is the 2n gamete formation mechanism involved in 'Fortune' and clementines. With SDR, PHR is positively linked with the distance of each locus to the centromere. With DD parents, PHR depends on the rate of preferential pairing and thus the proportion of disomic versus tetrasomic segregations. We have compared the genetic structure of two populations of diploid gametes of clementine (one population of 2n gametes and one of 2x gametes produced by DD) with SNP and SSR markers covering the 9 citrus chromosomes. The DD displays mostly tetrasomic segregation; however three linkage groups present intermediate segregation and one displays a tendency for disomy. The PHR in the 2n gametes is 2/3 of that obtained in 2x gametes produced by DD. The two methods of triploid production appear complementary in terms of genotypic variability. 4x X 2x hybridizations are more efficient than 2x X 2x for developing new cultivars phenotypically closer to the diploid parent of the DD. Conversely, 2x X 2x hybridisations provides a greater opportunity to select innovative products. (Resume)

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

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

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

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

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

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

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

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

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.

Adventitious embryony from nucellar cells is the mechanism leading to apomixis in citrus. However, singular cases of polyembryony have been reported in non-apomictic genotypes as a consequence of 2x×4x hybridisations and in vitro culture of isolated nucellus. The origin of the plants obtained as a consequence of these two processes is still unclear. In this work, we systematically analyzed the genetic structure (ploidy and allelic constitution at SSR locus) of plants obtained from polyembryonic seeds arising from 2x×4x sexual hybridisations or regenerated from nucellus culture in vitro of different non-apomictic citrus genotypes. Histological studies were also conduced to try to identify the initiation process of polyembryony in nonapomictic genotypes. We demonstrate that all plants obtained from the same undeveloped seed in 2x×4x hybridisations resulted from fission of the original zygotic embryo. Also, the plants obtained from in vitro culture of nucellus were recovered by somatic embryogenesis from cells having the same genotype as the zygotic embryos of the same seed. It appears that in non-apomictic citrus, proembryos or embryogenic cells are formed by fission of the original zygotic embryo and that the development of these adventitious embryos, normally hampered, can take place in vivo or in vitro as result of two different mechanisms that prevent the dominance of the initial zygotic embryo. (Texte integral)

Seedlessness is one of the most important characteristics for mandarins for the fresh-fruit market and mandarin triploid hybrids have this trait. Citrus triploid plants can be recovered by 2x x 2x sexual hybridisations as a consequence of the formation of unreduced gametes at low frequency. Triploid embryos are found in small seeds that do not germinate under greenhouse conditions. Extensive breeding programs based on this type of hybridisation require very effective methodologies for embryo rescue and ploidy evaluation. In this work, we describe an effective methodology to recover triploid hybrids from 2x x 2x hybridisations based on in vitro embryo rescue and ploidy level determination by means of flow cytometry. The influence of parents and environmental conditions on obtaining triploid hybrids has been analysed. The strongest effect was associated with the genotype of the female parent while a strong interaction was found between the male parent genotype and environmental conditions. The effect of the female parent genotype on the length of the juvenile phase was also demonstrated by observing a large number of progenies over the last 10 years. The methodology described here has enabled us to obtain over 4,000 triploid hybrids so far, of which 13 have been protected in the European Union and two are being extensively planted by citrus growers to establish new commercial plots. These triploid hybrids have been analysed with simple sequence repeats markers to differentiate all the new triploid varieties and their parents, and thus molecular identification will help defend plant breeders' rights.

International audience; 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.

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

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

Alternaria brown spot (ABS) in citrus is a serious disease caused by the tangerine pathotype of the fungus Alternaria alternate, 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. In the present work, we have taken advantage of the particular genetic structures of unreduced megagametophytes resulting from second division restitution (SDR) (leading to triploid hybrid recovery) to map a genome region linked to ABS resistance in citrus. 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 region linked to ABS resistance near the centromere of chromosome III. SSR and SNP markers were developed for efficient early marker-assisted selection (MAS) of ABS resistant hybrids. (Resume d'auteur)

Background Mapping centromere locations in plant species provides essential information for the analysis of genetic structures and population dynamics. The centromere’s position affects the distribution of crossovers along a chromosome and the parental heterozygosity restitution by 2n gametes is a direct function of the genetic distance to the centromere. Sexual polyploidisation is relatively frequent in Citrus species and is widely used to develop new seedless triploid cultivars. The study’s objectives were to (i) map the positions of the centromeres of the nine Citrus clementina chromosomes; (ii) analyse the crossover interference in unreduced gametes; and (iii) establish the pattern of genetic recombination in haploid clementine gametes along each chromosome and its relationship with the centromere location and distribution of genic sequences. Results Triploid progenies were derived from unreduced megagametophytes produced by second-division restitution. Centromere positions were mapped genetically for all linkage groups using half-tetrad analysis. Inference of the physical locations of centromeres revealed one acrocentric, four metacentric and four submetacentric chromosomes. Crossover interference was observed in unreduced gametes, with variation seen between chromosome arms. For haploid gametes, a strong decrease in the recombination rate occurred in centromeric and pericentromeric regions, which contained a low density of genic sequences. In chromosomes VIII and IX, these low recombination rates extended beyond the pericentromeric regions. The genomic region corresponding to a genetic distance

Despite potential benefits granted by genetically modified (GM) citrus trees, their release and commercialization raises concerns about their potential environmental impact. The transfer via pollen of transgenes to crosscompatible cultivars is deemed to be the greatest source for environmental exposure. In this work, three different citrus genotypes carrying the uidA (GUS) tracer marker gene (pollen donors) and a non-GM selfincompatible 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 floweringsynchronized 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, indicating that pollen competition strongly contributed to transgene confinement. This is the first study on transgene flow in citrus. It provide crucial information on the safety and field performance of GM citrus that can serve as a basis for further field trials and as a guide for (case-by-case) regulatory policies. (Resume d'auteur)

Mandarin genotypes can be split in two main groups: the acidic and the sweet mandarins. Commonly, acidic are used as rootstocks when sweet mandarins are the varieties that are consumed as fruits. The acidic 'Cleopatra' mandarin (Citrus reshni Hort. ex Tan.) is considered to be the most salt stress tolerant rootstock since it is able to limit the absorption of chloride and sodium at root level. As a consequence, limited amount of toxic ions are translocated from root to leaves. If salt stress tolerance of 'Cleopatra' mandarin and its hybrids has been well documented, little data exist for sweet mandarins. We investigated 16 genotypes representing a large diversity in mandarins subjected to salt stress. Physiological parameters such as stomatal conductance and leaf chloride contents were analyzed. If most of the acidic varieties were tolerant, also some sweet varieties presented traits of tolerance. Taking into account the results of leaf ion contents, we observed that large leaf chloride contents were not always associated with sensitivity. (Resume d'auteur)

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

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

A dinucleotide microsatellite-enriched genomic library was obtained from Carica papaya L. Sixty-nine microsatellites were evaluated on three genotypes of C. papaya and seven species of the neighbour genus Vasconcellea. Among them, 45 markers were characterized on a sample of 29 accessions of C. papaya from the Caribbean region and 11 accessions of Vasconcellea from the Andes of Colombia and Ecuador. Twenty-four gave polymorphism in C. papaya and only four among the seven species of Vasconcellea, confirming the divergence between both genera. (Resume d'auteur)

Citrus species are widely grown in the world. Plant characteristics of the rootstock populations used for orange, lemon, mandarin and grapefruit cultivars should be known as well as those of the cultivars that help true-to-type nursery plant production. In this study, cell nuclei were isolated from Icaf tissues of seedlings of trifoliate orange, sour orange, rough lemon, 'Volkamer' lemon, 'Cleopatra' mandarin, 'Hyokan', 'Sanbokan', 'Kinkoje', 'Carrizo' citrange and 'Swingle' citrumelo, and then fluorescence intensities were measured on propidium iodide-stained nuclei by flow cytometry. Nuclei isolated from the triploid 'Tahiti' lime with a known nuclear genome size were used as the internal standard to estimate the nuclear DNA content of Citrus seedling populations in absolute units. Results obtained from cytograms and histograms indicated that all seedlings analyzed were diploid. In addition, differences between the species for nuclear DNA content were also found to be significant. 'Hyokan' seedlings had the biggest genome size, 0.984 pg/2C, whereas trifoliate orange seedlings had the smallest genome size, 0.678 pg/2C. Flow cytometry analysis could be used for obtaining accurate and rapid results for cytological observations of seedling populations of Citrus. (Resume d'auteur)

Isozyme variation was studied in 87 plants from 32 cultivated and wild accessions of banana passion fruit (P. tripartita var. mollissima, P. tripartita var. tripartita, P. tarminiana, and P. mixta), rosy passion fruit (P. cumbalensis), tin-tin (P. pinnatistipula), gulian (P. ampullacea), P. antioquensis, P. bracteosa, and P. manicata, from the Andes of Venezuela, Colombia, and Ecuador. Six polymorphic enzyme systems (IDH, PGDH, PGM, DIA, PRX, and ACP) revealed 31 zymotypes characterized by the presence or absence of 31 electromorphs. Cluster analysis separated clearly the accessions of P. tarminiana, P. tripartita, P. mixta, and P. cumbalensis from the less typical species of subgenus Tacsonia, which is consistent with morphological evidence. P. mixta showed the highest intraspecific variation and the closest affinity with P. tripartita. The accessions of these two species formed two clusters, one dominated by Colombian genotypes and the other dominated by Ecuadorian genotypes. One of the P. tripartita var. mollissima accessions clustered close to P. tarminiana accessions. The affinity between these three species is particularly interesting for conservation and use of banana passion fruit genetic resources. All the other species formed monospecific clusters.

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

DNA variation was studied at the intra- andinterspecific levels within the genus Passiflora, with particular emphasis on the subgenusTacsonia and its relationship with the subgenusPassiflora. AFLP fingerprints were generated for35 accessions from 21 species, 14 of which belong to subgenusTacsonia, and four to subgenusPassiflora. The morphologically intermediateP. manicata (subgenusManicata) was also included, as wereP. trinervia (subgenusPsylanthus) and P.alnifolia (subgenus Decaloba). The two latter were used as outgroup species. Twoprimer combinations generated 34–80 scorable fragments pergenotype for a total of 260 fragments. Polymorphism analyses spannedthe levels from individual genotype to section. SubgenusTacsonia presented wide genetic variability atboth intra- and interspecific levels, particularly within wildspecies. The classification based on these markers generally agreeswell with current taxonomic definitions of common species, but lesswith definitions of sections. A geographical component was evident inthe intraspecific variation of P.mixta, P.tripartita var. mollissimaand P. tarminiana, withwider variation in Ecuador and Peru than in Colombia and Venezuela.These same three species, plus P.cumbalensis, formed adjacent clusters, showingcloser affinity with each other than with the other species ofsubgenus Tacsonia. As expected, P. manicata clustered at anintermediate position between subgenera Tacsoniaand Passiflora.

International audience; Different physiological behavior of a wide range of varieties and species belonging to the Citrus genus was analyzed when subjected to salt stress with the aim to seek new sources of tolerance that might be specie-specific. Our goal was to use physiological results obtained along a salt stress in order to clarify if it would be possible to associate them with the known citrus genetic diversity. For that purpose, we have selected 20 different genotypes representing the major species on the basis of the genetic diversity of Citrus genus complemented with one intergeneric hybrid Carrizo citrange (C. sinensis 9 P. trifoliata). A moderate salt stress of 75 mM of NaCl was applied for 12 weeks. For control plants, the main parameters contributing for more than 25 % to the diversity on the two axes of principal component analysis (PCA) were chlorophyll content, photosynthesis and Fv/ Fm under light. However, the dispersal of species and varieties on the PCA did not show any particular structure. Under salt stress condition, four parameters (leaf chloride content, leaf chlorophyll content, photosynthesis and stomatal conductance) contributed more specifically to the dispersion on PCA representation with more than 15 % of contribution for each parameter. Large differences were observed within citrus genus: mandarin and pummelo presented good tolerance to salt stress while citron was very sensitive. Furthermore, all secondary genotypes that presented good tolerance to salt tolerance shared mandarin or pummelo as female parent.

Cultivated citrus are selections from, or hybrids of, wild progenitor species whose identities and contributions to citrus domestication remain controversial. Here we sequence and compare citrus genomes-a high-quality reference haploid clementine genome and mandarin, pummelo, sweet-orange and sour-orange genomes- and show that cultivated types derive from two progenitor species. Although cultivated pummelos represent selections from one progenitor species, Citrus maxima, cultivated mandarins are introgressions of C. maxima into the ancestral mandarin species Citrus reticulata. The most widely cultivated citrus, sweet orange, is the offspring of previously admixed individuals, but sour orange is an F1 hybrid of pure C. maxima and C. reticulata parents, thus implying that wild mandarins were part of the early breeding germplasm. A Chinese wild 'mandarin' diverges substantially from C. reticulata, thus suggesting the possibility of other unrecognized wild citrus species. Understanding citrus phylogeny through genome analysis clarifies taxonomic relationships and facilitates sequence-directed genetic improvement., 栽培されている柑橘類は、野生祖先種から選択されたもの、またはその交雑種であるが、その祖先種の正体、および柑橘類栽培化に対する寄与には依然として議論がある。今回、柑橘類ゲノム（クレメンタインの高精度標準ハプロイドゲノム、ならびにマンダリン、パメロ、スイートオレンジ、およびサワーオレンジのゲノム）の配列解読および比較を行ったところ、栽培種が2つの祖先種に由来することが明らかにされた。栽培パメロは単一祖先種 Citrus maxima から選択されたことがわかったが、栽培マンダリンは祖先マンダリン種 Citrus reticulata に C. maxima から遺伝子移入が行われたものであった。最も広く栽培されている柑橘類であるスイートオレンジは過去に混種した個体の子孫であるが、サワーオレンジは純粋な C. maxima と C. reticulata とのF1雑種であり、このことは、野生のマンダリンが初期の育種生殖系列の要素であったことを意味している。中国の野生「マンダリン」は C. reticulata とは大きく異なっており、別の未確認の野生柑橘種である可能性が示唆される。ゲノム解析で柑橘類の系統発生を理解することにより、分類学的な関係が解明され、配列本位の遺伝的改良が促進される。

Iron is one of the most important elements in plant mineral nutrition. Fe deficiency is a critical abiotic stress factor for Mediterranean citriculture; the development of marker-assisted selection for this trait would greatly enhance rootstock breeding. In this study, DNA sequencing and single-stranded conformation polymorphism (SSCP) analyses were performed to determine the allelic diversity of genes associated with tolerance to iron chlorosis in citrus. Two candidate iron chlorosis tolerance genes were selected from existing Citrus EST databases and Arabidopsis thaliana genome databases. Ferritin-3 chloroplast precursor and putative membrane transporter candidate gene sequences were used to define primers in conserved regions. Six citrus genotypes from the basic taxon of Citrus were used to identify polymorphic regions in the genes. Direct sequencing of the amplified DNA fragments from the candidate genes was performed, and single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were identified after sequence alignment. Based on the DNA sequencing analysis, a total of 6840 nucleotides of DNA were sequenced to identify SNPs and indels. In total, 263 SNPs and 15 indels were identified for both genes. We detected 38.45 SNPs and 2.19 indels for each 1000 b on average from the DNA sequencing results. New primers were designed in conserved areas flanking polymorphic ones for SSCP analysis. SSCP-PCR analysis was performed with 25 citrus genotypes. The neighbor-joining method was used for cluster analysis. Trifoliate genotypes and their hybrids (known to be sensitive to iron chlorosis) clustered together, whereas genotypes tolerant to iron chlorosis were more spread out on the dendrogram. Mandarins also showed high diversity for both genes according to SSCP results. Differences were found among sour orange genotypes known to have differential tolerance behavior to iron chlorosis. Iron is one of the most important elements in plant mineral nutrition. Fe deficiency is a critical abiotic stress factor for Mediterranean citriculture; the development of marker-assisted selection for this trait would greatly enhance rootstock breeding. In this study, DNA sequencing and single-stranded conformation polymorphism (SSCP) analyses were performed to determine the allelic diversity of genes associated with tolerance to iron chlorosis in citrus. Two candidate iron chlorosis tolerance genes were selected from existing Citrus EST databases and Arabidopsis thaliana genome databases. Ferritin-3 chloroplast precursor and putative membrane transporter candidate gene sequences were used to define primers in conserved regions. Six citrus genotypes from the basic taxon of Citrus were used to identify polymorphic regions in the genes. Direct sequencing of the amplified DNA fragments from the candidate genes was performed, and single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were identified after sequence alignment. Based on the DNA sequencing analysis, a total of 6840 nucleotides of DNA were sequenced to identify SNPs and indels. In total, 263 SNPs and 15 indels were identified for both genes. We detected 38.45 SNPs and 2.19 indels for each 1000 b on average from the DNA sequencing results. New primers were designed in conserved areas flanking polymorphic ones for SSCP analysis. SSCP-PCR analysis was performed with 25 citrus genotypes. The neighbor-joining method was used for cluster analysis. Trifoliate genotypes and their hybrids (known to be sensitive to iron chlorosis) clustered together, whereas genotypes tolerant to iron chlorosis were more spread out on the dendrogram. Mandarins also showed high diversity for both genes according to SSCP results. Differences were found among sour orange genotypes known to have differential tolerance behavior to iron chlorosis.

Background The most economically important Citrus species originated by natural interspecific hybridization between four ancestral taxa (Citrus reticulata, Citrus maxima, Citrus medica, and Citrus micrantha) and from limited subsequent interspecific recombination as a result of apomixis and vegetative propagation. Such reticulate evolution coupled with vegetative propagation results in mosaic genomes with large chromosome fragments from the basic taxa in frequent interspecific heterozygosity. Modern breeding of these species is hampered by their complex heterozygous genomic structures that determine species phenotype and are broken by sexual hybridisation. Nevertheless, a large amount of diversity is present in the citrus gene pool, and breeding to allow inclusion of desirable traits is of paramount importance. However, the efficient mobilization of citrus biodiversity in innovative breeding schemes requires previous understanding of Citrus origins and genomic structures. Haplotyping of multiple gene fragments along the whole genome is a powerful approach to reveal the admixture genomic structure of current species and to resolve the evolutionary history of the gene pools. In this study, the efficiency of parallel sequencing with 454 methodology to decipher the hybrid structure of modern citrus species was assessed by analysis of 16 gene fragments on chromosome 2. Results 454 amplicon libraries were established using the Fluidigm array system for 48 genotypes and 16 gene fragments from chromosome 2. Haplotypes were established from the reads of each accession and phylogenetic analyses were performed using the haplotypic data for each gene fragment. The length of 454 reads and the level of differentiation between the ancestral taxa of modern citrus allowed efficient haplotype phylogenetic assignations for 12 of the 16 gene fragments. The analysis of the mixed genomic structure of modern species and cultivars (i) revealed C. maxima introgressions in modern mandarins, (ii) was consistent with previous hypotheses regarding the origin of secondary species, and (iii) provided a new picture of the evolution of chromosome 2. Conclusions 454 sequencing was an efficient strategy to establish haplotypes with significant phylogenetic assignations in Citrus, providing a new picture of the mixed structure on chromosome 2 in 48 citrus genotypes. Electronic supplementary material The online version of this article (doi:10.1186/s12863-014-0152-1) contains supplementary material, which is available to authorized users.

Citrus somatic hybridization and cybridization via protoplast fusion has become an integral part of citrus variety improvement programs worldwide. Citrus somatic hybrid plants have been regenerated from more than 200 parental combinations, and several cybrid combinations have also been produced. Applications of somatic hybridization to citrus scion improvement include the production of quality tetraploid breeding parents that can be used in interploid crosses to generate seedless triploids, and the direct production of triploids by haploid + diploid fusion. Applications of somatic hybridization to citrus rootstock improvement include the production of allotetraploid hybrids that combine complementary diploid rootstocks, and to combine citrus with sexually incompatible or difficult to hybridize genera that possess traits of interest for germplasm expansion. A few somatic hybrid tetraploid breeding parents have flowered, are fertile, and are being used as pollen parents to generate triploids. Several allotetraploid somatic hybrid rootstocks are performing well in commercial field trials, and show great promise for tree size control. Seed trees of most of these somatic hybrid rootstocks are producing adequate nucellar seed for standard propagation. Somatic hybridization is expected to have a positive impact on citrus cultivar improvement efforts.

Clausena excavata, a native of Southeast Asia, displays characteristics of disease tolerance and adaptability to different kinds of soils. This species contains potential parents for the improvement of Citrus fruit rootstocks. However, it is tetraploid, unlike most Citrus and related species, which are diploid. Bringing the ploidy level back to 2n=2x would allow us to consider integrating this species into the varietal creation programme by somatic hybridisation. A diplohaploidisation programme by anther culture is currently being conducted. The effects of the hormonal balance on androgenesis and on the maturation of androgenetic embryos are described. Thirty-six hormonal combinations using benzylamino purine (BAP) and dichlorophenoxyacetic acid (2,4-D) were examined. The tendency to embryogenesis varies from 0 to 9% depending on the hormonal concentration and combination. A total of 106 plants were regenerated, of which 91 were hexaploid, 11 were tetraploid, and only two were diploid after the flow cytometry evaluation. Histological study showed that the embryos were derived not from somatic embryogenesis of the mother tissues but from microspores. (Resume d'auteur)