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.

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.

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.

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.

Convergent evolution leading to the appearance of furanocoumarins in citrus plants.

Furanocoumarins are defense molecules mainly described in four plant families that are phylogenetically distant. Molecular characterization of the biosynthetic pathway has been started for many years in Apiaceae and Rutaceae. The results obtained thus far in Apiaceae indicated a major role of cytochromes P450 (P450s) in the CYP71 family. In the present work, we describe the importance of another subfamily of P450s, CYP82D, identified by using a deep analysis of the citrus (Rutaceae) genome and microarray database. CYP82D64 is able to hydroxylate xanthotoxin to generate 5-OH-xanthotoxin. Minor and limited amino acid changes in the CYP82D64 coding sequence between Citrus paradisi and Citrus hystrix provide the enzyme in the latter with the ability to hydroxylate herniarin, but with low efficiency. The kinetic constants of the enzyme are consistent with those of other enzymes of this type in plants and indicate that it may be the physiological substrate. The activity of the enzyme is identical to that of CYP71AZ6 identified in parsnip, showing possible evolutionary convergence between these two families of plants. It is highly possible that these molecules are derived from the synthesis of ubiquitous coumarins throughout the plant kingdom.

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.

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.

Noemi Controls Production of Flavonoid Pigments and Fruit Acidity and Illustrates the Domestication Routes of Modern Citrus Varieties.

In citrus, the production of anthocyanin pigments requires the activity of the transcriptional activator Ruby. Consequently, loss-of-function mutations in Ruby result in an anthocyaninless phenotype [1]. Several citrus accessions, however, have lost the ability to produce these pigments despite the presence of wild-type Ruby alleles. These specific mutants have captivated the interest of botanists and breeders for centuries because the lack of anthocyanins in young leaves and flowers is also associated with a lack of proanthocyanidins in seeds and, most notably, with an extreme reduction in fruit acidity (involving about a three-unit change in pH). These mutants have been defined collectively as "acidless" [2-4]. We have identified Noemi, which encodes a basic helix-loop-helix (bHLH) transcription factor and which controls these apparently unrelated processes. In accessions of Citron, limetta, sweet lime, lemon, and sweet orange, acidless phenotypes are associated with large deletions or insertions of retrotransposons in the Noemi gene. In two accessions of limetta, a change in the core promoter region of Noemi is associated with reduced expression and increased pH of juice, indicating that Noemi is a major determinant of fruit acidity. The characterization of the Noemi locus in a number of varieties of Citron indicates that one specific mutation is ancient. The presence of this allele in Chinese fingered Citrons and in those used in the Sukkot Jewish ritual [5] illuminates the path of domestication of Citron, the first citrus species to be cultivated in the Mediterranean. This allele has been inherited in Citron-derived hybrids with long histories of cultivation.

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

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

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.

A simple, fast and inexpensive method to assess salt stress tolerance of aerial plant part: Investigations in the mandarin group.

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.

The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways.

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.

Maximum-likelihood method identifies meiotic restitution mechanism from heterozygosity transmission of centromeric loci: application in citrus.

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.

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.

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.

Coumarin and furanocoumarin quantitation in citrus peel via ultraperformance liquid chromatography coupled with mass spectrometry (UPLC-MS).

Coumarins and furanocoumarins are secondary metabolites commonly found in citrus plants. These molecules are allelochemical compounds in plants that have controversial effects on humans, such as phototoxicity and the commonly described interactions with drugs, referred to as the "grapefruit juice effect". Thus, it is important to develop a reliable method to identify and quantitate the coumarins and furanocoumarins in citrus extracts. For this purpose, we herein describe an ultraperformance liquid chromatography coupled with mass spectrometry (UPLC-MS)-based method. We first developed a rapid UPLC method (20 min) to separate the isomers of each furanocoumarin. A subsequent single ion monitoring MS detection method was performed to distinguish between the molecules, which were possibly coeluting but had different molecular weights. The method was successfully used to separate and quantitate 6 coumarins and 21 furanocoumarins in variable amounts within peel extracts (flavedo and albedo) of 6 varieties of Citrus (sweet orange, lemon, grapefruit, bergamot, pummelo, and clementine). This method combines high selectivity and sensitivity in a rapid analysis and is useful for fingerprinting Citrus species via their coumarin and furanocoumarin contents.

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.

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.