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.

Cold-Pressing Olive Oil in the Presence of Cryomacerated Leaves of Olea or Citrus: Nutraceutical and Sensorial Features.

The nutraceutical properties of extra-virgin olive oil (EVOO) can be further improved by the addition of olive leaves during olive pressing. However, while Citrus leaves are rich sources of bioactive substances, no data are available in the literature about the effect of Citrus leaf addition on the nutraceutical and sensorial profiles of olive oil. This study aimed at comparing the chemical and sensorial qualities of olive oils obtained from ripe olives pressed together with either Olea or Citrus spp. (lemon or orange) cryomacerated leaves. General composition parameters as well as major antioxidants and antioxidant activity were measured. A panel test evaluation, as well as headspace volatile characterization (headspace solid phase microextraction, HS-SPME), were also performed. All data were compared with an EVOO extracted from the same olive batch used as control. It was possible to obtain Leaf Olive Oils (LOOs) characterized by a higher (p < 0.05) content of antioxidants, compared to the control sample, and the highest oleuropein concentration was detected in the olive oil extracted in presence of olive leaf (+50% in comparison with the control). All the LOOs showed a higher smell complexity and the scent of ripe fruit was generally mitigated. Lemon and olive LOOs showed the best smell profile.

Phylogenetic origin of limes and lemons revealed by cytoplasmic and nuclear markers.

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. reticulate 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.

Short- and long-term effects of carbohydrate limitation on sugar and organic acid accumulation during mandarin fruit growth.

BACKGROUND: The physiological roles of organic acids in fruit cells are not fully understood, especially in citrus, whereas the decline in titratable acidity during ripening shown by many citrus fruits is due to the utilization of citric acid. We induced carbohydrate depletion by removing source leaves at two key periods in mandarin development (early and full citric acid accumulation). Then, we assessed the resulting changes in the short term (within 48 h) and long term (several weeks until ripening). RESULTS: Control mature fruits were characterized by elevated fresh weight, large diameters and high quantities of malic acid, citric acid and sucrose. At the same stage, fruits subjected to early or late defoliation had higher glucose, fructose, citric acid concentrations and lower sucrose concentrations. They differed only in their malic acid concentrations, which were higher in early defoliation fruits and similar in late defoliation fruits when compared to control fruits. Finally, fruits subjected to late defoliation were characterized by high proline and γ-aminobutyric acid concentrations, and low fructose and glucose concentrations. CONCLUSION: We have shown that short- and long-term carbohydrate limitation modifies sugar and organic acid metabolism during mandarin fruit growth. © 2015 Society of Chemical Industry.

Next generation haplotyping to decipher nuclear genomic interspecific admixture in Citrus species: analysis of chromosome 2.

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.

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 nuclear phylogenetic analysis: SNPs, indels and SSRs deliver new insights into the relationships in the 'true citrus fruit trees' group (Citrinae, Rutaceae) and the origin of cultivated species.

BACKGROUND AND AIMS: Despite differences in morphology, the genera representing 'true citrus fruit trees' are sexually compatible, and their phylogenetic relationships remain unclear. Most of the important commercial 'species' of Citrus are believed to be of interspecific origin. By studying polymorphisms of 27 nuclear genes, the average molecular differentiation between species was estimated and some phylogenetic relationships between 'true citrus fruit trees' were clarified. METHODS: Sanger sequencing of PCR-amplified fragments from 18 genes involved in metabolite biosynthesis pathways and nine putative genes for salt tolerance was performed for 45 genotypes of Citrus and relatives of Citrus to mine single nucleotide polymorphisms (SNPs) and indel polymorphisms. Fifty nuclear simple sequence repeats (SSRs) were also analysed. KEY RESULTS: A total of 16 238 kb of DNA was sequenced for each genotype, and 1097 single nucleotide polymorphisms (SNPs) and 50 indels were identified. These polymorphisms were more valuable than SSRs for inter-taxon differentiation. Nuclear phylogenetic analysis revealed that Citrus reticulata and Fortunella form a cluster that is differentiated from the clade that includes three other basic taxa of cultivated citrus (C. maxima, C. medica and C. micrantha). These results confirm the taxonomic subdivision between the subgenera Metacitrus and Archicitrus. A few genes displayed positive selection patterns within or between species, but most of them displayed neutral patterns. The phylogenetic inheritance patterns of the analysed genes were inferred for commercial Citrus spp. CONCLUSIONS: Numerous molecular polymorphisms (SNPs and indels), which are potentially useful for the analysis of interspecific genetic structures, have been identified. The nuclear phylogenetic network for Citrus and its sexually compatible relatives was consistent with the geographical origins of these genera. The positive selection observed for a few genes will help further works to analyse the molecular basis of the variability of the associated traits. This study presents new insights into the origin of C. sinensis.

Correspondence between the Compositional and Aromatic Diversity of Leaf and Fruit Essential Oils and the Pomological Diversity of 43 Sweet Oranges (Citrus x aurantium var sinensis L.).

Orange (Citrus x aurantium var sinensis) is the most widely consumed citrus fruit, and its essential oil, which is made from the peel, is the most widely used in the food, perfume, and cosmetics industries. This citrus fruit is an interspecific hybrid that would have appeared long before our era and would result from two natural crosses between mandarin and pummelo hybrids. This single initial genotype was multiplied by apomictic reproduction and diversified by mutations to produce hundreds of cultivars selected by men essentially based on phenotypic characteristics of appearance, spread of maturity, and taste. Our study aimed to assess the diversity of essential oil composition and variability in the aroma profile of 43 orange cultivars representing all morphotypes. In agreement with the mutation-based evolution of orange trees, the genetic variability tested with 10 SSR genetic markers was null. The oils from peels and leaves extracted by hydrodistillation were analyzed for composition by GC (FID) and GC/MS and for aroma profile by the CATA (Check All That Apply) method by panelists. Oil yield varied between varieties by a factor of 3 for PEO and a factor of 14 for LEO between maximum and minimum. The composition of the oils was very similar between cultivars and was mainly dominated by limonene (>90%). However, small variations were observed as well as in the aromatic profile, with some varieties clearly distinguishing themselves from the others. This low chemical diversity contrasts with the pomological diversity, suggesting that aromatic variability has never been a selection criterion in orange trees.

GABA shunt pathway is stimulated in response to early defoliation-induced carbohydrate limitation in Mandarin fruits.

The regulation of sugar and organic acid metabolism during fruit development has a major effect on high-quality fruit production. The reduction of leaf area is a common feature in plant growth, induced by abiotic and biotic stresses and disturbing source/sink ratio, thus impacting fruit quality. Here, we induced carbohydrate limitation by partial leaf defoliation at the beginning of the second stage of mandarin development (before the citrate peak). Resulting changes were monitored in the short-term (48 h and 1 week) and long-term (7 weeks) after the defoliation. Short-term response to early defoliation implied metabolic settings to re-feed TCA for sustaining respiration rate. These features involved (i) vacuolar sucrose degradation (high acid invertase activity and mRNA expression level) and enhanced glycolytic flux (high ATP-phosphofructokinase activity), (ii) malic and citric acid utilization (increased phosphoenolpyruvate kinase and NADP-Isocitrate dehydrogenase) associated with vacuolar citric acid release (high mRNA expression of the transporter CsCit1) and (iii) stimulation of GABA shunt pathway (low GABA content and increased mRNA expression of succinate semialdehyde dehydrogenase). A steady-state proline level was found in ED fruits although an increase in P5CS mRNA expression level. These results contribute to a better knowledge of the molecular basis of the relationship between defoliation and sugar and organic acid metabolism in mandarin fruit.

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

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

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.

Comparative use of InDel and SSR markers in deciphering the interspecific structure of cultivated citrus genetic diversity: a perspective for genetic association studies.

Genetic stratification associated with domestication history is a key parameter for estimating the pertinence of genetic association study within a gene pool. Previous molecular and phenotypic studies have shown that most of the diversity of cultivated citrus results from recombination between three main species: C. medica (citron), C. reticulata (mandarin) and C. maxima (pummelo). However, the precise contribution of each of these basic species to the genomes of secondary cultivated species, such as C. sinensis (sweet orange), C. limon (lemon), C. aurantium (sour orange), C. paradisi (grapefruit) and recent hybrids is unknown. Our study focused on: (1) the development of insertion-deletion (InDel) markers and their comparison with SSR markers for use in genetic diversity and phylogenetic studies; (2) the analysis of the contributions of basic taxa to the genomes of secondary species and modern cultivars and (3) the description of the organisation of the Citrus gene pool, to evaluate how genetic association studies should be done at the cultivated Citrus gene pool level. InDel markers appear to be better phylogenetic markers for tracing the contributions of the three ancestral species, whereas SSR markers are more useful for intraspecific diversity analysis. Most of the genetic organisation of the Citrus gene pool is related to the differentiation between C. reticulata, C. maxima and C. medica. High and generalised LD was observed, probably due to the initial differentiation between the basic species and a limited number of interspecific recombinations. This structure precludes association genetic studies at the genus level without developing additional recombinant populations from interspecific hybrids. Association genetic studies should also be affordable at intraspecific level in a less structured pool such as C. reticulata.

Investigations of the Chemical Composition and Aromatic Properties of Peel Essential Oils throughout the Complete Phase of Fruit Development for Two Cultivars of Sweet Orange (Citrus sinensis (L.) Osb.).

The peel essential oil (PEO) of sweet orange is used for flavoring liquors or foods and in the perfumery and cosmetics industry. The fruit maturity stage can modify the essential oil composition and aromatic properties, but little information is available on the evolution of PEO during the entire time set of fruit development. In this study, the yield, chemical composition and aromatic profile over the three phases of orange development were monitored. Four fruit traits (peel color, weight, acidity and sweetness) were recorded to characterize fruit development. Fruits of two sweet orange cultivars were sampled every two weeks from June to May of the next year. PEO was obtained by hydrodistillation and analyzed by gas chromatography coupled with a flame ionization detector (GC-FID). Compounds were identified with GC coupled with mass spectrometry (GC/MS). Ten expert panelists using the descriptor intensity method described the aromatic profile of PEO samples. The PEO composition was richer in oxygenated compounds at early fruit development stages, with an aromatic profile presenting greener notes. During fruit growth (Phases I and II), limonene's proportion increased considerably as a few aliphatic aldehydes brought the characteristic of orange aroma. During fruit maturation (from November to March), the PEO composition and aromatic profile were relatively stable. Later, some modifications were observed. Regardless of the fruit development stage, the two sweet oranges presented distinct PEO compositions and aromatic profiles. These results constitute a temporal reference for the chemical and aromatic evolution of sweet orange PEO in the fruit development process under Mediterranean conditions. During the first two phases of fruit development, many changes occur in the PEO composition and aroma, suggesting that their exploitation could create new products.

Distinguishing citrus varieties based on genetic and compositional analyses.

Simple sequence repeats (SSR) markers and secondary metabolite composition were used in combination to study seven varieties of citrus for the first time. With reference to established accessions of citrus, two of the varieties (Chanh Giay and Ma Nao Pan) were predicted to be Mexican key limes, while three were mandarin hybrids (Nagpur, Pontianak and Dalandan) and the remaining two (Qicheng and Mosambi) were related to the sweet orange. Notably, Dalandan was genetically more like a mandarin despite often referred to as an orange locally, whereas Mosambi was more likely to be a sweet orange hybrid although it has also been called a sweet lime due to its green peel and small size. Several key secondary metabolites such as polymethoxyflavones (sinensetin, tangeretin etc.), furanocoumarins (bergapten, citropten etc.) and volatiles (citronellol, α-sinensal etc.) were identified to be potential biomarkers for separation of citrus species. However, despite having similar genetic profiles, variations in the volatile profile of the two limes were observed; similarly, there were differences in the secondary metabolite profiles of the three mandarin hybrids despite having a common ancestral parent, highlighting the usefulness of genetic and compositional analyses in combination for revealing both origins and flavour profiles especially in citrus hybrids. This knowledge would be crucial for variety screening and selection for use in flavour or fragrance creation and application.

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

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

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

The Papeda Citrus subgenus includes several species belonging to two genetically distinct groups, containing mostly little-exploited wild forms of citrus. However, little is known about the potentially large and novel aromatic diversity contained in these wild citruses. In this study, we characterized and compared the essential oils obtained from peels and leaves from representatives of both Papeda groups, and three related hybrids. Using a combination of GC, GC-MS, and 13C-NMR spectrometry, we identified a total of 60 compounds in peel oils (PO), and 76 compounds in leaf oils (LO). Limonene was the major component in almost all citrus PO, except for C. micrantha and C. hystrix, where ß-pinene dominated (around 35%). LO composition was more variable, with different major compounds among almost all samples, except for two citrus pairs: C. micrantha/C. hystrix and two accessions of C. ichangensis. In hybrid relatives, the profiles were largely consistent with their Citrus/Papeda parental lineage. This high chemical diversity, not only among the sections of the subgenus Papeda, but also between species and even at the intraspecific level, suggests that Papeda may be an important source of aroma diversity for future experimental crosses with field crop species.

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

Ichang lemon is a citrus fruit whose rind gives off a delicious and much appreciated fragrance and flavor. The volatile components of the fruit peel of Ichang lemon were investigated by GC-MS and GC-O (AEDA method). Simultaneously, its genetic origin was identified by using diagnostic SNP markers specific to ancestral species and multiallelic SSR and InDel markers. Ichang lemon combines three ancestral genomes (Citrus maxima, Citrus ichangensis, and Citrus reticulata) and may be a pummelo × Yuzu hybrid. Although the major compounds of the Ichang lemon aromatic profile were present in Citrus junos, a few pummelo-specific compounds were also detected, such as indole and nootkatone, in agreement with its maternal lineage. 3-Methyl-3-sulfanylbutyl acetate, reported to occur in passion fruit and brewed coffee, was identified by GC-MS, GC-QTOF-MS, and GC-FTIR for the first time in citrus. This odor-active compound has a sulfurous, tropical fruity, green note.

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

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

Karyological Analysis and DNA Barcoding of Pompia Citron: A First Step toward the Identification of Its Relatives.

Pompia is a citrus fruit endemic of Sardinia, Italy, with an essential oil profile showing outstanding anti-inflammatory and anti-microbic properties. Despite its remarkable pharmaceutical potential, little taxonomic and genetic information is available for this species. We applied flow cytometry and classical cytogenetic techniques to assess the DNA content and to reconstruct the karyotype of several Pompia accessions. Molecular data from plastid DNA barcoding and nuclear DNA sequencing were used to study the genetic distance between Pompia and other citrus species. Flow cytometric estimates of DNA content and somatic chromosome counts suggest that Pompia is a regular diploid Citrus species. DNA polymorphisms of nuclear and chloroplast markers allowed us to investigate the genetic relationships between Pompia accessions and other Citrus species. Based on DNA polymorphism data we propose that Pompia is a very recent interspecific hybrid generated by a cross between C. aurantium (as seed bearer) and C. medica (as pollen donor). Our findings pave the way for further and more specific investigations of local Pompia germplasm resources that may help the preservation and valorisation of this valuable citrus fruit tree.

Nutrient Deficiency Tolerance in Citrus Is Dependent on Genotype or Ploidy Level.

Plants require essential minerals for their growth and development that are mainly acquired from soil by their roots. Nutrient deficiency is an environmental stress that can seriously affect fruit production and quality. In citrus crops, rootstock/scion combinations are frequently employed to enhance tolerance to various abiotic stresses. These tolerances can be improved in doubled diploid genotypes. The aim of this work was to compare the impact of nutrient deficiency on the physiological and biochemical response of diploid (2x) and doubled diploid (4x) citrus seedlings: Volkamer lemon, Trifoliate orange × Cleopatra mandarin hybrid, Carrizo citrange, Citrumelo 4475. Flhorag1 (Poncirus trifoliata + and willow leaf mandarin), an allotetraploid somatic hybrid, was also included in this study. Our results showed that depending on the genotype, macronutrient and micronutrient deficiency affected certain physiological traits and oxidative metabolism differently. Tetraploid genotypes, mainly Flhorag1 and Citrumelo 4475, appeared resistant compared to the other genotypes as indicated by the lesser decrease in photosynthetic parameters (P net, F v/F m, and G s) and the lower accumulation of oxidative markers (MDA and H2O2) in roots and leaves, especially after long-term nutrient deficiency. Their higher tolerance to nutrient deficiency could be explained by better activation of their antioxidant system. For the other genotypes, tetraploidization did not induce greater tolerance to nutrient deficiency.