Karyological analysis and DNA barcoding of pompia citron: A first step toward the identification of its relatives

open7Pompia 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.openViglietti G.; Galla G.; Porceddu A.; Barcaccia G.; Curk F.; Luro F.; Scarpa G.M.Viglietti, G.; Galla, G.; Porceddu, A.; Barcaccia, G.; Curk, F.; Luro, F.; Scarpa, G. M

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 x 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

Acta Horticulturae

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

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

International audienceIchang 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 x 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 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. ConclusionsCitrus medica contributed to all limes and lemons and was the direct male parent for the main sub-groups in combination with C. micrantha or close papeda species (for C. aurata, C. excelsa, C. macrophylla and C. aurantifolia – ‘Mexican’ lime types of Tanaka’s taxa), C. reticulata (for C. limonia, C. karna and C. jambhiri varieties of Tanaka’s taxa, including popular citrus rootstocks such as ‘Rangpur’ lime, ‘Volkamer’ and ‘Rough’ lemons), C. aurantium (for C. limetta and C. limon – yellow lemon types – varieties of Tanaka’s taxa) or the C. maxima × C. reticulata hybrid (for C. limettioides – ‘Palestine sweet’ lime types – and C. meyeri). Among triploid limes, C. latifolia accessions (‘Tahiti’ and ‘Persian’ lime types) result from the fertilization of a haploid ovule of C. limon by a diploid gamete of C. aurantifolia, while C. aurantifolia triploid accessions (‘Tanepao’ lime types and ‘Madagascar’ lemon) probably result from an interspecific backcross (a diploid ovule of C. aurantifolia fertilized by C. medica). As limes and lemons were vegetatively propagated (apomixis, horticultural practices) the intra-sub-group phenotypic diversity results from asexual variations

Genetic diversity revealed by physiological behavior of citrus genotypes subjected to salt stress

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

Autotetraploid trifoliate orange (Poncirus trifoliata) rootstocks do not impact clementine quality but reduce fruit yields and highly modify rootstock/scion physiology

Two autotetraploid (4x) trifoliate orange (Poncirus trifoliata (L) Rat.) rootstocks and the corresponding diploid (2 x) rootstocks were compared for their impact on clementine "Commune" (Citrus clementina Hort. ex Tan) production and on rootstock/scion physiology. Cumulative fruit yield, fruit quality (sugars, organic acids, carotenoids, hesperidin) and scion physiology (stem growth, leaf starch, sugars, chlorophyll content, phenolic content and gas exchange parameters) were investigated. The cumulative fruit yield over 11 years was lower in 4x rootstock/clementine associations and correlated with reduced vigor of the clementine scion. Fruit quality criteria were not affected by the ploidy level of the rootstocks, with the exception of hesperidin content, which was higher with 4x than 2x rootstock/clementine associations. Leaf chlorophyll and phenolic contents were higher with 4x than 2x rootstock/clementine associations. In leaves, the rate of photosynthesis and stomatal conductance was much higher with 2x rootstocks; however, the maximum electron transport rate was lower when compared to the 4x rootstocks. Similarly, leaf starch and sugar contents were much higher with 4x than 2x. Taken together, these results demonstrate that the use of 4x rootstocks dramatically changes both tree physiology and fruit yield, without promoting large changes in fruit quality criteria. (C) 2011 Elsevier B.V. All rights reserved

Acta Horticulturae

Rootstock provides better adaptation to biotic and abiotic constraints. Poncirus and its hybrids are widely used since they are tolerant to Citrus tristeza virus. However, when grafted, most of them are considered to be sensitive to salinity and water deficit. Diversity does exist within the Poncirus trifoliata species, but little is known regarding the behaviour variability of genotypes to abiotic constraints. The citrus diversity of 72 P. trifoliata genotypes was investigated using SSR markers. Two major genetic groups were clearly identified. Five genotypes, belonging to each genetic group, were then selected to investigate their properties of tolerance to water deficit. Water deficit was applied by withdrawing irrigation for 4 weeks. Physiological parameters such as leaf stomatal conductance and gravitational water quantity were estimated. Among genotypes, we observed that some genotypes such as 'Rubidoux' were clearly more tolerant to water deficit than others such as 'Pomeroy'. Interestingly, the genotypes that were sensitive belonged to one genetic group and the less tolerant ones belonged to the other group. Therefore, our results suggest that among the Poncirus genus, it is possible to select genotypes as parent in breeding programs, which are better tolerant to water deficit

Sensitivity to high salinity in tetraploid citrus seedlings increases with water availability and correlates with expression of candidate genes

We investigated tolerance to high salinity in well-irrigated diploid and tetraploid citrus. Comparisons were made between two diploids (2x) of trifoliate orange (Poncirus trifoliata (L.) Raf.) and willow leaf mandarin (Citrus deliciosa Ten), their respective doubled diploids (4x) and the allotetraploid (FLHORAG1) obtained from the protoplast fusion of trifoliate orange and Willow leaf mandarin. Salinity stress was applied by progressively increasing the concentration of NaCl from 50 mM to 400 mM for 8 weeks. Two-year-old plants were watered daily. Maximum quantum yield of PSII, and leaf and root chloride and sodium content were monitored. We previously reported that under moderate saline stress, citrus 4x genotypes were more tolerant that the 2x, but under these experimental conditions, 4x seedlings were certainly more sensitive to salt stress than 2x, as they accumulated more toxic ions and were more affected than 2x. Chloride accumulation in 4x leaves was greater and the maximum quantum yield of PSII was more reduced in 4x than in 2x. The expression of several candidate genes involved in signal transduction, sodium and chloride transport, osmotic adjustment, regulation of the stomata opening and detoxification processes were also investigated by quantitative real-time reverse transcription-PCR. A high correlation was observed between phenotype of sensitivity to stress and gene expression changes