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.

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.

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.

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.

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.

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

Modern cultivated Citrus species and varieties result from interspecific hybridization between four ancestral taxa. Among them, Citrus maxima and Citrus reticulata, closely associated with the pummelo and mandarin horticultural groups, respectively, were particularly important as the progenitors of sour and sweet oranges (Citrus aurantium and Citrus sinensis), grapefruits (Citrus paradisi), and hybrid types resulting from modern breeding programs (tangors, tangelos, and orangelos). The differentiation between the four ancestral taxa and the phylogenomic structure of modern varieties widely drive the phenotypic diversity's organization. In particular, strong phenotypic differences exist in the coloration and sweetness and represent important criteria for breeders. In this context, focusing on the genes of the sugar, carotenoid, and chlorophyll biosynthesis pathways, the aim of this work was to develop a set of diagnostic single-nucleotide polymorphism (SNP) markers to distinguish the ancestral haplotypes of C. maxima and C. reticulata and to provide information at the intraspecific diversity level (within C. reticulata or C. maxima). In silico analysis allowed the identification of 3,347 SNPs from selected genes. Among them, 1,024 were detected as potential differentiation markers between C. reticulata and C. maxima. A total of 115 SNPs were successfully developed using a competitive PCR technology. Their transferability among all Citrus species and the true citrus genera was very good, with only 0.87% of missing data. The ancestral alleles of the SNPs were identified, and we validated the usefulness of the developed markers for tracing the ancestral haplotype in large germplasm collections and sexually recombined progeny issued from the C. reticulata/C. maxima admixture gene pool. These markers will pave the way for targeted association studies based on ancestral haplotypes.

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

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

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.

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.

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.

Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones.

Nutrient deficiency has economic and ecological repercussions for citrus fruit crops worldwide. Citrus crops rely on fertilization to maintain good fruit output and quality, whereas new crop management policy aims to reduce fertilizers input. New rootstocks are needed to meet to this constraint, and the use of new tetraploid rootstocks better adapted to lower nutrient intake could offer a promising way forward. Here we compared physiological, biochemical and anatomic traits of leaves in diploid (2x) and doubled-diploid (4x) Citrumelo 4475 (Citrus paradisi L. Macf. × Poncirus trifoliata L. Raf.) and Volkamer lemon (Citrus limonia Osb.) seedlings over 7 months of nutrient deficiency. Photosynthetic parameters (Pnet, Gs and Fv/Fm) decreased, but to a lesser extent in 4x genotypes than 2x. Degradation of the ultrastructural organelles (chloroplasts and mitochondria) and compound cells (thylakoids and starches) was also lower in 4x genotypes, suggesting that tetraploidy may enhance tolerance to nutrient deficiency. However, leaf surface (stomata, stomatal density and epithelial cells) showed no nutrient deficiency-induced change. In 4x Citrumelo 4475, the higher tolerance to nutrient deficiency was associated with a lower MDA and H2O2 accumulation than in the 2x, suggesting a more efficient antioxidant system in the 4x genotype. However, few differences in antioxidant system and oxidative status were observed between 2x and 4x Volkamer lemons.

Genotyping by sequencing reveals the interspecific C. maxima / C. reticulata admixture along the genomes of modern citrus varieties of mandarins, tangors, tangelos, orangelos and grapefruits.

The mandarin horticultural group is an important component of world citrus production for the fresh fruit market. This group formerly classified as C. reticulata is highly polymorphic and recent molecular studies have suggested that numerous cultivated mandarins were introgressed by C. maxima (the pummelos). C. maxima and C. reticulata are also the ancestors of sweet and sour oranges, grapefruit, and therefore of all the "small citrus" modern varieties (mandarins, tangors, tangelos) derived from sexual hybridization between these horticultural groups. Recently, NGS technologies have greatly modified how plant evolution and genomic structure are analyzed, moving from phylogenetics to phylogenomics. The objective of this work was to develop a workflow for phylogenomic inference from Genotyping By Sequencing (GBS) data and to analyze the interspecific admixture along the nine citrus chromosomes for horticultural groups and recent varieties resulting from the combination of the C. reticulata and C. maxima gene pools. A GBS library was established from 55 citrus varieties, using the ApekI restriction enzyme and selective PCR to improve the read depth. Diagnostic polymorphisms (DPs) of C. reticulata/C. maxima differentiation were identified and used to decipher the phylogenomic structure of the 55 varieties. The GBS approach was powerful and revealed 30,289 SNPs and 8,794 Indels with 12.6% of missing data. 11,133 DPs were selected covering the nine chromosomes with a higher density in genic regions. GBS combined with the detection of DPs was powerful for deciphering the "phylogenomic karyotypes" of cultivars derived from admixture of the two ancestral species after a limited number of interspecific recombinations. All the mandarins, mandarin hybrids, tangelos and tangors analyzed displayed introgression of C. maxima in different parts of the genome. C. reticulata/C. maxima admixture should be a major component of the high phenotypic variability of this germplasm opening up the way for association studies based on phylogenomics.

A L-type lectin gene is involved in the response to hormonal treatment and water deficit in Volkamer lemon.

Combination of biotic and abiotic stress is a major challenge for crop and fruit production. Thus, identification of genes involved in cross-response to abiotic and biotic stress is of great importance for breeding superior genotypes. Lectins are glycan-binding proteins with a functions in the developmental processes as well as in the response to biotic and abiotic stress. In this work, a lectin like gene, namely ClLectin1, was characterized in Volkamer lemon and its expression was studied in plants exposed to either water stress, hormonal elicitors (JA, SA, ABA) or wounding to understand whether this gene may have a function in the response to multiple stress combination. Results showed that ClLectin1 has 100% homology with a L-type lectin gene from C. sinensis and the in silico study of the 5'UTR region showed the presence of cis-responsive elements to SA, DRE2 and ABA. ClLectin1 was rapidly induced by hormonal treatments and wounding, at local and systemic levels, suggesting an involvement in defence signalling pathways and a possible role as fast detection biomarker of biotic stress. On the other hand, the induction of ClLectin1 by water stress pointed out a role of the gene in the response to drought. The simultaneous response of ClLectin1 expression to water stress and SA treatment could be further investigated to assess whether a moderate drought stress may be useful to improve citrus performance by stimulating the SA-dependent response to biotic stress.

Tetraploid Carrizo citrange rootstock (Citrus sinensis Osb.×Poncirus trifoliata L. Raf.) enhances natural chilling stress tolerance of common clementine (Citrus clementina Hort. ex Tan).

Low temperatures can disturb the development, growth and geographic distribution of plants, particularly cold-sensitive plants in the Mediterranean area, where temperatures can reach seasonally low levels. In citrus crops, scion/rootstock combinations are used to improve fruit production and quality, and increase tolerance to biotic and abiotic stresses. In the last decade, several studies have shown that tetraploid citrus seedlings or rootstocks are more tolerant to abiotic stress than their respective diploid. The objective of this study was to test whether the use of tetraploid rootstocks can improve the chilling tolerance of the scion. We compared physiological and biochemical responses to low seasonal temperatures of common Clementine (Citrus sinensis Osb.×Poncirus trifoliata L. Raf.) grafted on diploid and tetraploid Carrizo citrange rootstocks, named C/2xCC and C/4xCC, respectively. During the coldest months, C/4xCC showed a smaller decrease in net photosynthesis (Pn), stomatal conductance (Gs), chlorophyll fluorescence (Fv/Fm), and starch levels, and lower levels of malondialdehyde and electrolyte leakage than C/2xCC. Specific activities of catalase (CAT), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) were higher in C/4xCC during the cold period, whereas chlorophyll, proline, ascorbate and hydrogen peroxide (H2O2) levels and superoxide dismutase (SOD) activity did not vary significantly between C/4xCC and C/2xCC throughout the study period. Taken together, these results demonstrate that tetraploid Carrizo citrange rootstock improves the chilling tolerance of common clementine (scion) thanks to a part of the antioxidant system.

Integrated Analysis by GC(RI), GC-MS and 1C NMR of Fortunella japonica Leaf Volatiles Obtained by Hydrodistillation, Microwave- assisted Hydrodistillation and Hydrolate Extraction.

The chemical composition of the essential oil (EO), microwave extract (ME) and hydrolate extract (HE) from the same batch of leaves of Fortunella japonica, was investigated: by combination of chromatographic (GC, CC) and spectroscopic techniques (GC-MS, 13C NMR). F. japonica essential oil and extracts are complex mixtures of 28-60 compounds being mainly oxygenated sesquiterpenes. The EO composition was dominated by germacrene D (14.9%), ß-elemol (9.1%), cis-guai-6-en-10ß-ol (6.3%), ß-eudesmol (5.5%), and δ-elemene (5.2%). Limonene was the unique monoterpene identified at appreciable amount (7.1%). The extract obtained by microwave assisted hydrodistillation contained as main components: ß-elemol (12.4%), germacrene D (9.9%), cis-guai-6-en- 10ß-ol (9.0%), ß-eudesmol (8.2%), germacra-l(l0),5-dien-4α-ol (7.1%) and α-eudesmol (6.4%). Finally, the highest content of oxygenated sesquiterpenes (near 92%) was found in the hydrolate extract displaying cryptomeridiol (23.3%, but totally absent in the EO and ME), ß-eudesmol (20.6%) and α-eudesmol (10.7%). Combined analysis by chromatographic and spectroscopic techniques appeared useful for identification of various sesquiterpenols bearing a tertiary alcohol function.

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.