New insights on limes and lemons origin from nuclear and cytoplasmic markers genenotyping and targeted nuclear gene sequencing

It is believed that Citrus medica, C. maxima, C. reticulate and C. micrantha have generated all cultivated citrus species. Depending on the classification, lemons and limes are classified either into two species, C. limon and C. aurantifolia (Swingle and Reece, 1967) or into more than 30 (Tanaka, 1977). In order to study the molecular phylogeny of this citrus group, we analyzed 23 targeted sequenced nuclear genes and used three mitochondrial and five chloroplastic markers for 15 lemons and limes compared with representatives of the four basic taxa. We observed three main groups, each one derived from direct interspecific hybridizations: (1) the Mexican lime group (C. aurantifolia), including C. macrophylla, arising from hybridizations between papeda (C. micrantha) and citron (C. medica); (2) the yellow lemon group (C. limon) that are hybrids between sour orange (C. aurantium, which is believed to be a hybrid between C. maxima and C. reticulata) and citron; and (3) a rootstock lemon/lime group (Rough lemon and Rangpur lime) that are hybrids between an acid mandarin and a citron. We also identified different probable backcrosses and genotypes with more complex origin. None of the analyzed limes and lemons shared the C. medica cytoplasm, while this taxon is the common nuclear contributor of all limes and lemons. Limes and lemons appear to be a very complex citrus varietal group with the contribution of the 4 basic taxa. Neither the Swingle and Reece classification nor the Tanaka fit with the genetic evidence. (Résumé d'auteur

Analysis of leaves chloride accumulation and phenotypic behavior of nine trifoliate citrus rootstocks under salt stress conditions : [P218]

Citrus in Morocco is mainly grafted on sour orange (Citrus aurantium L.) rootstock which should be abandoned due to its susceptibility to citrus tristeza virus (CTV). Salinity also becomes a serious problem in citrus growing areas of Morocco. The choice of the right rootstock is the most practical means to overcome the problem. Poncirus trifoliata (L.) Raf. and several of its hybrids (citrange, citrumello, citrandarin) are known to be tolerant to CTV. The purpose of the present work was to evaluate nine trifoliate citrus rootstocks, introduced in 1996 from San Giuliano Inra-Cirad Agronomic Research Station, Corsica France, for their behavior against salt stress. Seedlings of nine citrus rootstocks were evaluated for their leaf chloride accumulation under salt conditions. This study was carried out in greenhouse conditions at Inra Experimental station El Menzeh, Morecco. Three-month-old plants were grown. in 0.5 liter containers of sand and peat moss in proportion of 2/3 or 1/3 (v/v), and irrigated four times per week and fertilized with complete nutritional solution. NaCI at 0 (control), 2 and 5 g/l was added to the watering solution for 2 additional months. Ali rootstocks showed specific symptoms of toxicity when irrigated with NaCI solution. The severity of symptoms increased with time and salt concentration and varied between rootstocks. Diverse effects of salt stress on stem. leaves and roots fresh and dry weights were also observed according to the rootstock genotype. Rootstock leaf analysis revealed that accumulation of chloride, varied in relation with the rootstocks genotype and salt concentration. The relations between the different parameters analyzed are discussed and a standardized method for hybrids population screening proposed. Among the nine trifoliate hybrids tested Citrauge C35 (ICVN 0110177), Citnunelo Sacaton (ICVN 0110144) and Troyer citrange (ICVN 0110197) have been identified, in our experimental conditions as the less sensitive to salt stress. (Texte intégral

Multilocus haplotyping by parallel sequencing to decipher the interspecific mosaic genome structure of cultivated citrus

The most important economic Citrus species originated from natural interspecific hybridization between four ancestral taxa (C. reticulata, C. maxima, C. medica and C. micrantha) with limited further interspecific recombination due to apomixis and vegetative propagation. Such reticulate evolution coupled with vegetative propagation results in genomes that are mosaics of large chromosome fragments of the basic taxa, in frequent interspecific heterozygosity. Breeding of these species is hampered by their complex heterozygous genomic structures. Haplotyping of multiple gene fragments along the genome should be a powerful approach to resolve the evolutionary history of the gene pools, to reveal the admixture genomic structure of current species and to develop innovative breeding schemes. We have analysed the efficiency of parallel sequencing with 454 methodology to decipher the hybrid structure of modern citrus species and cultivars along chromosome 2. Four hundred fifty four amplicon libraries were established with the fluidigm array system for 48 genotypes and 16 gene fragments of chromosome 2. Haplotypes were established from the reads of each accession and phylogenetic analyses were performed from the haplotypic data of 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 hypothesis regarding the origin of secondary species; and (iii) provided a new picture of the evolution of chromosome 2. Perspectives to rebuild the main secondary species from the basic taxa are discussed. (Résumé d'auteur

Comparative values of SSRs, SNPs and InDels for citrus genetic diversity analysis

SSRs have long been considered as almost ideal markers for genetic diversity analysis. With the increasing availability of sequencing data, SNPs and InDels become major classes of codominant markers with genome wide coverage. We have analyzed the respective values of SSRs, InDels, and SNPs for intra and interspecific Citrus genetic diversity analysis. Moreover, we have compared the diversity structure revealed by markers mined in a single heterozygous genotype (the clementine) and markers mined in a large interspecific survey. A random set of 25 markers was selected for each marker class to genotype 48 citrus accessions. SSRs were the most polymorphic markers at the intraspecific level allowing complete varietal differentiation within basic taxa (Citrus reticulata, Citrus maxima, Citrus medica). However, SSRs gave the lowest values for interspecific differentiation, followed by SNPs and InDels, that displayed low intraspecific variability but high interspecific differentiation. A clear effect of the discovery panel was observed for SNPs and InDels. The ascertainment biases associated with the clementine heterozygosity mining resulted mainly in an over estimation of within C. reticulata diversity and an underestimation of the interspecific differentiation. Therefore SSRs are very useful for intraspecific structure analysis while SNPs and InDels mined in large discovery panel will be more powerful to decipher the interspecific mosaic structure of secondary cultivated species. (Résumé d'auteur

Genetic structure and phylogeny of the 'true citrus fruit trees' group (Citrinae, Rutaceae)

Despite considerable differences in morphology, the general representing 'true citrus fruit trees' are sexually compatible, but their phylogenetic relationships remain unclear. Most of the important commercial species of Citrus are believed to be of interspecific origin. By studying SNP and indel polymorphisms of 27 nuclear genes on 32 genotypes of Citrus and relatives of Citrus, the phylogenetic relationship between 'true citrus fruit trees' was clarified. Additionally, four mitochondrial (mtDNA) and five chloroplastic (cpDNA) SSRs markers were analysed for the citrus species to specify the maternal phylogeny of Citrus secondary species. A total of 16238 kb of DNA was sequenced for each genotype, and 1156 SNPs and 58 indels were identified. Nuclear phylogenetic analysis revealed that C. reticulata, Poncirus, and Fortunella form a clade that is clearly differentiated from the clade that includes three other basic taxa of cultivated citrus (C. maxima, C. medica and C. micrantha). Clymenia is included in a third strong clade with Eremocitrus and Microcitrus. The nuclear phylogeny of Citrus and its sexually compatible relatives was consistent with the geographic origins of these genera. The origin of Citrus secondary species was analysed both at nuclear and cytoplasmic level and the conclusions mostly agree with previous hypotheses. The SNPs and indels identified at the nuclear level from a relatively large discovery panel will be useful for systematic characterisation of citrus germplasm, both at intra- and inter-specific level. (Résumé d'auteur

A phylogenomic study based on Genotyping By Sequencing unravels the interspecific mosaic structures of the cultivated Citrus genomes

Reticulate evolution, including hybrid speciation, introgression and lateral gene transfer is frequentin plant species. When coupled with vegetative propagation, it results in mosaic genomes of large genomic fragments, from different species or sub species. These complex inter(sub)-‐specific genomic structures can support a major part of the phenotypic diversity organization. Cultivated Citrus are a good example of such genepool issued from reticulate evolution with limited number of further interspecific recombination. It is generally agreed that four ancestral taxa (Citrus maxima: pummelos, Citrus reticulata: mandarins, Citrus medica: citrons and Citrus micrantha: papedas) are the ancestors of all the cultivated citrus species. These four species, have undergone allopatric evolution. This has led to a strong genetic differentiation which is also found for many phenotypic characters. The so-­‐called secondary species (sweet and sour oranges, grapefruits, lemons and limes) are the result of a reticulate evolution between these four ancestral taxa. Facultative apomixis (nucellar polyembrionny) then limited the number of interspecific meiosis cycle. The aplication of NGS on reduced genome representation with methods such as GBS (Genotyping By Sequencing) coupled with the availability of a clementine reference sequence open the way for pangenomic studies of large populations. The objective of this work was to validate a GBS approach on citrus in order to identify a pangenomic panel of diagnostic markers (DSNPs) of each ancestral taxa and to decipher the phylogenomic structures of 56 citrus varieties representative of the ancestral taxa and secondary species. The DSNP panel was also used to analyse the phylogenomic structures of diploid and triploid recombining populations of the Cirad-­‐Inra breeding programs. GBS library were prepared with ApekI and a selective PCR to improve the depth of the analysis. 56 accessions were pooled and sequenced in one line of Illumina HISEQ-­‐2000 (single reads). Diversity structure analysis showed that the varieties are distributed among the four ancestral taxa in perfect consistency with the previous studies carried out with SSRs, Indels and SNPs markers. The GBS approach is thus validated. 14926 DSNPs were identified. These diagnostic markers allowed to infer efficiently the majority of the phylogenomic karyotypes of the 56 Citrus accessions and revealed the interspecific recombination point in the diploid hybrids of breeding populations. For polyploid germplasm and hybrids, needing the evaluation of allelic doses, we were not able to infer these doses from the relative reads number at individual locus level. The potential of analysis at genome fragment level, covering numerous DSNPs, is under study. This work demonstrate the potential of GBS for deciphering the phylogenomic structure of the modern citrus varieties and recent hybrids of breeding populations. It bring new insights on the origins of citrus fruit and open the way for genetic associations studies and QTLs analysis based on phylogenomics and further to genomic selection

Local Ancestry lnference approaches to unravel plant genome mosaic: a simulation-based evaluation

Hybridization events between species and subspecies are considered as major evolutionary steps, possibly contributing to the advent of new phenotypes. These events are widespread in several crop species and are expected to produce genomes with a mosaic structure of sequence blocks originating from different ancestry. With the development of NGS genotyping technologies, several population genomics approaches have been proposed to infer the ancestry of genome segments, by comparing polymorphism patterns across individuals along chromosomes. However, these Local Ancestry lnference (LAI) methods have mainly been developed for applications in animal models, and human most particularly. They are based on assumptions which do not always fit plant models due to more complex genome structures (e.g. different ploidy levels, variable heterozygosity levels within species) or different reproductive systems (e.g. vegetative propagation, selfing). ln this context. there is a need to evaluate available methods on plant models. To that end, we developed a small and flexible R tool ta simulate mosaic genome data under a wide variety of scenarios representative of plant model characteristics. We evaluated two main types of LAI methods i) exploratory approaches (based on multivariate analysis), and ii) full probabilistic approaches (based on Hidden Markov Models) that are able ta use unphased genotypic data and handle more than two ancestries. The results will be presented and discussed

Le germoplasme agrumes de San Giuliano : une base pour la production de plants CAC et certifiés

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