François Luro

New universal mitochondrial PCR markers reveal new information on maternal citrus phylogeny

The aim of this work was to provide a set of mitochondrial markers to reveal polymorphism and to study the maternal phylogeny in citrus. We first used 44 universal markers previously described in the literature: nine of these markers produced amplification products but only one revealed polymorphism in citrus. We then designed six conserved pairs of primers using the complete mitochondrial DNA sequences of Arabidopsis thaliana and Beta vulgaris to amplify polymorphic intergenic and intronic regions. From these six pairs of primers, three from introns of genes coding for NADH dehydrogenase subunits 2, 5, and 7, revealed polymorphism in citrus. First, we confirmed that citrus have a maternal mitochondrial inheritance in two populations of 250 and 120 individuals. We then conducted a phylogenic study using four polymorphic primers on 77 genotypes representing the diversity of Citrus and two related genera. Seven mitotypes were identified. Six mitotypes (Poncirus, Fortunella, Citrus medica, Citrus micrantha, Citrus reticulata, and Citrus maxima) were congruent with previous taxonomic investigations. The seventh mitotype enabled us to distinguish an acidic mandarin group (‘Cleopatra’, ‘Sunki’ and ‘Shekwasha’) from other mandarins and revealed a maternal relationship with Citrus limonia (‘Rangpur’ lime, ‘Volkamer’ lemon) and Citrus jambhiri (‘Rough’ lemon). This mitotype contained only cultivated species used as rootstocks due to their good tolerances to abiotic stress. Our results also suggest that two species classified by Swingle and Reece, Citrus limon, and Citrus aurantifolia, have multiple maternal cytoplasmic origins.

A reference genetic map of C. clementina hort. ex Tan.; citrus evolution inferences from comparative mapping

BackgroundMost 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.ResultsFive 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.ConclusionsA 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.

Characterization of microsatellite markers in mandarin orange (Citrus reticulata Blanco)

A dinucleotide‐enriched genomic library was obtained from mandarin orange (Citrus reticulata Blanco). A subset of 101 positive clones was sequenced and primers were designed. The loci were screened for levels of variation using 26–29 wild mandarin oranges collected in Vietnam. Forty‐three loci were polymorphic with the number of alleles ranging from two to 18. The observed heterozygosity (HO) and expected heterozygosity (HE) were from 0.03 to 0.96 and from 0.03 to 0.92, respectively.

Non-random inheritance of mitochondrial genomes in Citrus hybrids produced by protoplast fusion

Abstract. Somatic hybridization offers the possibility of manipulating chloroplast and mitochondrial genomes and evaluating their role on cultivar qualities in citrus. Numerous associations between Willow-leaf mandarin (Citrus deliciosa Ten.), as embryogenic parent, and sweet orange cv. Valencia (Citrus sinensis (L.) Osb.), as mesophyll parent, and between Willow-leaf mandarin (embryogenic parent) and grapefruit cv. Duncan (Citrus paradisi Macf.) (mesophyll parent) were obtained by the fusion of protoplasts induced by polyethylene glycol. Regenerated plants were characterized by flow cytometry and nuclear and mitochondrial DNA restriction fragment length polymorphism (RFLP). All plants were diploid. Diploid plants with the nuclear RFLP patterns of mandarin or sweet orange were identified in the progeny between these two parents, while only grapefruit nuclear types were found in the mandarin + grapefruit progeny. The diploid plants with the nuclear profile of the mesophyll parent originated systematically from cells formed through spontaneous association of the nuclear genome of the mesophyll parent and the mitochondrial genome of the embryogenic parent. These plants are assumed to be alloplasmic hybrids or cybrids. They were viable and have been propagated for field testing.

Comparative analysis of proteome changes induced by the two spotted spider mite Tetranychus urticae and methyl jasmonate in citrus leaves.

Citrus plants are currently facing biotic and abiotic stresses. Therefore, the characterization of molecular traits involved in the response mechanisms to stress could facilitate selection of resistant varieties. Although large cDNA microarray profiling has been generated in citrus tissues, the available protein expression data are scarce. In this study, to identify differentially expressed proteins in Citrus clementina leaves after infestation by the two-spotted spider mite Tetranychus urticae, a proteome comparison was undertaken using two-dimensional gel electrophoresis. The citrus leaf proteome profile was also compared with that of leaves treated over 0-72h with methyl jasmonate, a compound playing a key role in the defense mechanisms of plants to insect/arthropod attack. Significant variations were observed for 110 protein spots after spider mite infestation and 67 protein spots after MeJA treatments. Of these, 50 proteins were successfully identified by liquid chromatography-mass spectrometry-tandem mass spectrometry. The majority constituted photosynthesis- and metabolism-related proteins. Five were oxidative stress associated enzymes, including phospholipid glutathione peroxidase, a salt stressed associated protein, ascorbate peroxidase and Mn-superoxide dismutase. Seven were defense-related proteins, such as the pathogenesis-related acidic chitinase, the protease inhibitor miraculin-like protein, and a lectin-like protein. This is the first report of differentially regulated proteins after T. urticae attack and exogenous MeJA application in citrus leaves.

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 (2×) of trifoliate orange (Poncirus trifoliata (L.) Raf.) and willow leaf mandarin (Citrus deliciosa Ten), their respective doubled diploids (4×) 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 4× genotypes were more tolerant that the 2×, but under these experimental conditions, 4× seedlings were certainly more sensitive to salt stress than 2×, as they accumulated more toxic ions and were more affected than 2×. Chloride accumulation in 4× leaves was greater and the maximum quantum yield of PSII was more reduced in 4× than in 2×. 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.

Genetic and chemical diversity of citron (Citrus medica L.) based on nuclear and cytoplasmic markers and leaf essential oil composition.

Native to southeast Asia, the citron (Citrus medica L.) was the first citrus fruit to be introduced to the Mediterranean area, in the third century BC, and remained its only citrus representative until the tenth century. The citron was used for its aroma - stemming from its essential oils in leaves and fruit peels - and as symbols in the Jewish religion. Subsequently, the cultivation of citron was extended significantly, peaking in the nineteenth century, when its fruits were used in cosmetics and confectioneries. The objective of this study was to examine the genetic diversity of the Mediterranean citron with regard to the multiplication and dissemination practices that were related to its uses. We studied the polymorphisms of 27 nuclear and cytoplasmic genetic markers of 24 citron varieties, preserved in the citrus germplasm of INRA-CIRAD, San Giuliano, France. The composition of leaf essential oils was determined to establish varieties and phylogenic relationships between accessions. Other major citrus species were included in the molecular analysis, which demonstrated the existence of 13 genetically linked citrons, differing from other citrus species, based on low heterozygosity and specific alleles; these citrons were considered true-type citrons, confirmed by their convergent chemical profiles. We also detected a polymorphism in the chloroplastic genome in these 13 citrons, which, when combined with allelic diversity of 2.4 alleles per locus, suggests that multiple citrons were introduced to the Mediterranean area in last 2 millennia. We determined the genetic origin and relationships of several varieties, such as Corsican, which could have arisen from the selfing of Poncire Commun. We noted a higher-than-expected polymorphism rate among Mediterranean citron varieties, likely due to crossfecundation. The chemical leaf oil composition of several economical varieties, such as Corsican, is distinct and can increase the quality of specific agriculture products for the cosmetics and candy industries.

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

BackgroundThe 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.Results454 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.Conclusions454 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.

Genetic diversity and population structure analysis of mandarin germplasm by nuclear, chloroplastic and mitochondrial markers

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