Development of SSR markers from Citrus clementina (Rutaceae) BAC end sequences and interspecific transferability In Citrus (P148)

Microsatellite primers were developed from bacterial artificial chromosome (BAC) end sequences (BES) of Citrus clementina and their transferability and polymorphism tested in the genus Citrus for future anchorage of physical and genetic maps and comparative interspecific genetic mapping. Using PAGE and DNA silver staining, 79 primer pairs were selected for their transferability and polymorphism among 526 microsatellites mined in BES. A preliminary diversity study in Citrus was conducted with 18 of them, in C. reticulata , C. maxima , C. medica , C. sinensis , C. aurantium , C. paradisi , C. lemon , C. aurantifolia, and some papedas (wild citrus), using a capillary electrophoresis fragment analyzer. Intra and interspecific polymorphism was observed, and heterozygous markers were identified for the different genotypes to be used for genetic mapping. These results indicate the utility of the developed primers for comparative mapping studies and the integration of physical and genetic maps. (Résumé d'auteur

Characterization of water deficit tolerance of Poncirus trifoliata genotypes related to diversity

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 specie 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 mayor 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 leaf stomatal conductance and quantum yield of PSII electron transport, soil water potential, leaf osmotic pressure and transpiration 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 tolerant ones belonged to the other group. Therefore, it sounds that among the Poncirus genus, it is possible to select genotypes as parent in breeding programs, which are more tolerant to water deficit. (Résumé d'auteur

Salt stress tolerance in acidic and sweet mandarins

Mandarin genotypes can be split in two main groups: the acidic and the sweet mandarins. Commonly, acidic are used as rootstocks when sweet mandarins are the varieties that are consumed as fruits. The acidic 'Cleopatra' mandarin (Citrus reshni Hort. ex Tan.) is considered to be the most salt stress tolerant rootstock since it is able to limit the absorption of chloride and sodium at root level. As a consequence, limited amount of toxic ions are translocated from root to leaves. If salt stress tolerance of 'Cleopatra' mandarin and its hybrids has been well documented, little data exist for sweet mandarins. We investigated 16 genotypes representing a large diversity in mandarins subjected to salt stress. Physiological parameters such as stomatal conductance and leaf chloride contents were analyzed. If most of the acidic varieties were tolerant, also some sweet varieties presented traits of tolerance. Taking into account the results of leaf ion contents, we observed that large leaf chloride contents were not always associated with sensitivity. (Résumé d'auteur

Physiological and molecular responses of diploid and tetraploid Carrizo Citrange under water Stress : S02P14

In citrus, the use rootstock promotes productivity, improves fruit quality and may confer resistance or tolerance to biotic and abiotic stress. 'Carrizo' citrange (Citrus sinensis [L.] Osbeck × Poncirus trifoliata [L.] Raf), is one of the most popular rootstock in the Mediterranean basin. It is sensitive to drought and salt stress but confers tolerance to Tristeza virus, and promotes very good fruit quality. Previous studies have shown that doubled diploid (4x) 'Rangpur' lime (Citrus limonia, Osbeck) seedlings are more tolerant to water deficit than their respective diploid (2x). In the present work, we have characterized the water deficit tolerance in 2x and 4x 'Carrizo' citrange seedlings. Water deficit was applied for 35 days, followed by irrigation. Several physiological parameters were measured periodically during the experiment and samples were collected to investigate i) the activity of enzymes involved in detoxification processes, ii) the expression analysis of candidate genes involved in ABA biosynthesis, as well as iii) ABA and H2O2 production. Doubled diploid 'Carrizo' citrange seedlings were showed to be more drought tolerant than 2x. Water deficit caused a greater reduction in photosynthetic rates and stomatal conductance in 2x compared to 4x. Also higher ABA and H2O2 production were induced in 2x when compared to 4x. The better tolerance of 4x seedlings is discussed to the light of candidate genes expression analysis and activities of enzymes of detoxification. Work supported by CNPq and CAPES. (Texte intégral

Citrus tetraploid rootstocks are more tolerant to salt stress than diploid

Salt stress has a dramatic impact on the citrus industry by decreasing the growth of trees and fruit yield. We studied at the physiological and molecular level diploid and tetraploid citrus rootstocks when confronted to salt stress. Those tetraploid plants originate from the chromosome duplication in nucellar cells (somatic cells) of the apomictic diploid parent. Diploid and autotetraploid of Poncirus trifoliate (Pomeroy Poncirus trifoliata) and WillowLeaf mandarin (Citrus deliciosa) were studied. The allotetraploid somatic hybrid FLHORAG1 which was obtained by electrofusion between Willow-Leaf mandarin and Pomeroy Poncirus protoplasts was as weil investigated. Poncirus trifoliate is known to be a salt sensitive rootstock. Willow leaf mandarin is not used as a rootstock but was supposed to have a greater property of salt tolerance, similar to other mandarins such as Cleopatra mandarin. The anatomy of the leaf of diploid and tetraploid plants was first investigated. The stomatal area of tetraploid is 1.6 fold higher than the one of diploid plants. Stomatal density was also different between diploid and tetraploid. A 50 mM sodium chloride (NaCl) stress was perforrned for 9 weeks. Salinity caused leaf injuries and leaf fall in diploid of Poncirus while the tetraploid plants were not damaged. Autotetraploids and the allotetraploid FLHORAG1 acted like salt tolerant when compared to diploid plants. Sodium and chloride accumulation were similar for both diploid and tetraploid genotypes suggesting that root exclusion and/or accumulation of toxic ions in vacuoles is not the only way for salt tolerance. In order to characterize the molecular deterrninants mediating the salt tolerance of polyploids we investigated the gene expression profiles by using cDNA-AFLP technique. Contrasted gene expression profiles were detected between diploids and tetraploids as weil as between control and stressed plants. Transcript Derived Fragments (TDFs) from contrasted profiles pattern were sequenced. 14 genes involved in osmotic adjustment, defense and signal transduction were isolated. The expression of sorne of those genes such as genes coding for the choline monooxigenase enzyme and the Group 5 late embryogenesis abundant protein (LEA5) and genes involved in the Abscissic Acid biosynthesis pathway are actually monitored by using Real Time PCR. (Texte intégral

Physiological responses of diploid and doubled diploid 'Rangpur' lime under water deficit

In citrus, the use of rootstocks promotes productivity, improves fruit quality and may confer resistance or tolerance to biotic and abiotic stress. 'Rangpur' lime (Citrus limonia, Osbeck) is one of the most tolerant rootstock to drought and is largely used by the Brazilian citrus industry. Previous studies have shown that, when grafted with a variety citrus doubled diploid (4x) 'Rangpur' lime rootstock is more tolerant to drought stress than the respective diploid (2x). In the present study, we characterized the water deficit tolerance in 2x and 4x 'Rangpur' lime seedlings. Water deficit was applied respectively for 21 days, followed by recovery irrigation. Physiological and biochemical parameters were measured periodically during the experiment and samples were collected for biochemical analysis. Doubled diploid seedlings were showed to be more water deficit tolerant than 2x. Water deficit caused a greater reduction in stomatal conductance in 2x compared to 4x. The 21st day of stress, leaf water potential in 2x was lower. Analyses of the activity of enzymes involved in detoxification process suggest that the better tolerance of 4x genotypes may be related to better scavenging of reactive oxygen species. (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

Autotetraploid Citrus limonia rootstocks are more tolerant to water deficit than parentalal diploids : [S1-2]

Water shortage of soils is one of the main abiotic constraints affecting growth and yield in citrus. When grown in the field, tetraploid seedlings and varieties grafted on tetraploid rootstocks have been shown to be slow growing plants. In this work, we investigated the anatomy and morphology of root and leaf diploid and autotetraploid Rangpur lime (Citrus limonia) seedlings grown in control condition. The autotetraploid line arose from chromosome doubling in nucellar cells of diploid Rangpur lime and has strictly the same allelic composition than the diploid one. In autotetraploids, roots and leaves were thicker and cell size was bigger than in diploids. Leaf stomatal conductance of autotetraploids was lower than for diploids. Using 20 K cDNA microarrays, leaf gene expression was investigated in both genotypes. A very limited number of genes were significantly differentially expressed in both genotypes (? 0.5%) suggesting that gene dosage per cell or post-transcriptional events may explain the phenotypic differentiation between diploids and autotetraploids. We also investigated the tolerance to water stress of diploid and autotetraploid seedlings and also their behaviour as rootstocks of Valencia Delta orange (Citrus sinensis) and citron (Citrus medica) varieties. At the beginning of the stress, leaf stomatal conductances of autotetraploid seedlings and varieties grafted on autotetraploid rootstocks were respectively lower than those of diploid seedlings and varieties grafted on diploid rootstocks. At the end of the experiment, autotetraploid seedlings and varieties grafted on autotetraploids showed the highest tolerances. This work suggests that greater tolerance is linked to a more efficient regulation of gas exchanges in autotetraploid seedlings and varieties grown on autotetraploids genotypes. Investigations of ABA root content in diploids and autotetraploids suggest that constitutive biosynthesis of this hormone is higher in autotetraploids. Analyses of candidate gene expression were performed at the root level. The results showed that NCED1, which is involved in the last step of ABA biosynthesis was over expressed in roots of autotetraploids. To have a better understanding of the impact of diploid and tetraploid rootstocks on scion, we have also investigated gene expression using microarrays and qRT-PCR in Valencia Delta leaves grafted on diploid and autotetraploid rootstocks in control and water deficit conditions. (Texte intégral

Genome sequence comparisons reveal interspecific hybridizations and admixture in citrus domestication : [W317]

Citrus is the most globally significant tree fruit, but its history of domestication is not well understood. Cultivated citrus types are believed to be direct selections from, or hybrids of, several progenitor species; however, the identities of these species and their contributions to modern cultivars remain unclear and controversial. A collection of different genomes of mandarins, pummelos, and oranges, including a high quality reference sequence from a haploid Clementine mandarin, has been assembled. By comparative analyses we show that these cultivated types were derived from two progenitor species, C. maxima and C. reticulata. Although it was determined that the sequenced cultivated pummelos represent selections from C. maxima, the cultivated mandarins proved to be introgressions of C. maxima into a distinct second population identified as the ancestral wild mandarin species, C. reticulata. Sweet and sour oranges were found to be hybrids between these two species. Sweet orange, the most widely cultivated citrus, arose as the offspring of previously admixed individuals. Sour (or Seville) orange was found to be an F1 hybrid of pure C. maximaand C. reticulata parents, indicating that wild mandarins were at least a part of the early breeding germplasm. Sequence analysis also revealed that at least one Chinese "wild mandarin" actually represents a citrus species distinct from C. reticulata, suggesting the possibility that other unrecognized wild citrus species may be identified. Understanding the phylogeny of cultivated citrus by comparative genome analysis will clarify taxonomic relationships and potentially lead to previously inconceivable opportunities for genetic improvement. (Texte intégral

International effort toward a SSR-based linkage map for C. clementina : [P128]

Following the difficulties encountered for assembling a 1.2 x sequencing of the highly heterozygous sweet orange genome, the International Citrus Genomic Consortium (ICOC) decided to estab1ish the first reference sequence of a whole nuclear citrus genome from a haploid Clementine. A saturated genetic linkage map of Clementine based on sequence-characterized markers was considered by the ICGC as an important too1 for genome sequence assemb1y. In this framework, CIRAD proposed to use an interspecific population C. maxima x C. clementina to implement the reference Clementine genetic map. A population of 250 hybrids of Chandler pummelo x Clementine was established in Corsica and 190 hybrids were used in this first phase of mapping. Collaboration was established between two French organizations (CIRAD and INRA), two groups from United States (UF-CREC and UCR), one Spanish institute (IVIA), INRA Morocco and Cukurova University from Turkey. Forty markers were found heterozygous in Clementine among a previous set of 90 SSR markers developed by CIRAD from microsatellite-enriched genomic libraries. With the objective to integrate the physical and genetic maps of Clementine, CIRAD and IVIA have developed new SSR markers from microsatellite sequences identified in BAC End Sequences (BES) of diploid Clementine. On hundred and 10 of these new markers were found heterozygous for Clementine or Chandler pummelo and were used for genotyping. INRA France deve1oped 500 SSR markers from ESTs databases and found 170 markers heterozygous for Clementine. INRA Morocco contributed to the genotyping of 112 SSR markers developed from EST databases and genomic libraries, while 50 ESTs SSR were analysed by Cukurova University. SSR markers mainly developed from EST databases and already mapped for sweet orange were genotyped by UF-CREC (70 markers) and UCR (60 markers) to allow comparisons among the C. sinensis. C. maxima and C. c1ementina maps. lndeed, taking advantage of the important allelic differentiation between Clementine and Chandler, two parallel linkage maps can be developed from this population. As perspective, in the framework of the global haploid Clementine sequencing project, a collaboration between the French and Spanish groups plans: (i) to extend the population size to 380 hybrids between Clementine and pummelo. and (ii) to develop an array from SNPs identified in Clementine BES for High- Throughput Genotyping. All genotyping data will be stored in the online TropGene database (http://tropgenedb.cirad.fr/). Additional international groups are very welcome to join the project, using these progenies for genotyping their own markers. This should contribute to a very high density map of Clementine and to comparative mapping studies between citrus species. (Texte intégral