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L W Timmer - One of the best experts on this subject based on the ideXlab platform.
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colonization of citrus leaves and secondary conidiation response to citrus flower extracts by non postbloom fruit drop strains of colletotrichum acutatum
Tropical Plant Pathology, 2010Co-Authors: S J Mackenzie, Natalia A Peres, L W TimmerAbstract:Postbloom fruit drop (PFD) is caused by a unique strain of Colletotrichum acutatum that infects citrus flowers, producing necrotic lesions on petals and the abscission of fruitlets. This strain survives on vegetative tissues during non-flowering periods, and conidial production on colonized tissue is stimulated by flower extracts. Genetically distinct strains from other hosts can cause PFD symptoms, but their survival on vegetative tissues and ability to sporulate in response to flower extracts is unknown. Isolates from anthracnose-affected blueberry, leatherleaf fern, strawberry, Key Lime and PFD-affected sweet orange were evaluated for their ability to survive on leaves after inoculation and to produce conidia in response to flower extracts. Viable propagules were recovered from leaves inoculated with all non-PFD isolates and recovery of some was equal to or greater than that of the PFD isolate. The number of propagules recovered increased for all isolates after treatment with flower extracts and was comparable across isolates. Application of sucrose to leaves inoculated with the PFD isolate increased the number of propagules recovered from leaves, suggesting that the response to flower extracts was nutritional. Non-PFD isolates of C. acutatum can colonize and reproduce on leaves, and their inability to cause epidemics is probably due to their reduced pathogenicity to flowers.
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host range and genetic relatedness of colletotrichum acutatum isolates from fruit crops and leatherleaf fern in florida
Phytopathology, 2009Co-Authors: S J Mackenzie, Natalia A Peres, M P Barquero, L F Arauz, L W TimmerAbstract:Isolates of Colletotrichum acutatum were collected from anthracnose-affected strawberry, leatherleaf fern, and Key Lime; ripe-rot-affected blueberry; and postbloom fruit drop (PFD)-affected sweet orange in Florida. Additional isolates from ripe-rot-affected blueberry were collected from Georgia and North Carolina and from anthracnose-affected leatherleaf fern in Costa Rica. Pathogenicity tests on blueberry and strawberry fruit; foliage of Key Lime, leatherleaf fern, and strawberry; and citrus flowers showed that isolates were highly pathogenic to their host of origin. Isolates were not pathogenic on foliage of heterologous hosts; however, several nonhomologous isolates were mildly or moderately pathogenic to citrus flowers and blueberry isolates were pathogenic to strawberry fruit. Based on sequence data from the internal transcribed spacer (ITS)1-5.8S rRNA-ITS2 region of the rDNA repeat, the glutaraldehyde-3-phosphate dehydrogenase intron 2 (G3PD), and the glutamine synthase intron 2 (GS), isolates from the same host were identical or very similar to each other and distinct from those isolated from other hosts. Isolates from leatherleaf fern in Florida were the only exception. Among these isolates, there were two distinct G3PD and GS sequences that occurred in three of four possible combinations. Only one of these combinations occurred in Costa Rica. Although maximum parsimony trees constructed from genomic regions individually displayed little or no homoplasy, there was a lack of concordance among genealogies that was consistent with a history of recombination. This lack of concordance was particularly evident within a clade containing PFD, Key Lime, and leatherleaf fern isolates. Overall, the data indicated that it is unlikely that a pathogenic strain from one of the hosts examined would move to another of these hosts and produce an epidemic.
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postbloom fruit drop of citrus and Key Lime anthracnose are caused by distinct phylogenetic lineages of colletotrichum acutatum
Phytopathology, 2008Co-Authors: Natalia A Peres, S J Mackenzie, Tobin L Peever, L W TimmerAbstract:Colletotrichum acutatum causes two diseases of citrus, postbloom fruit drop (PFD) and Key Lime anthracnose (KLA). PFD is a disease restricted to flowers of sweet orange and most other citrus, and symptoms include petal necrosis, abscission of developing fruit, and the formation of persistent calyces. KLA is a disease of foliage, flowers, and fruits of Key Lime only, and symptoms include necrotic lesions on leaves, fruits, twigs, flowers, and blight of entire shoots. The internal transcribed spacers 1 and 2 and the gene encoding the 5.8S ribosomal RNA subunit within the nuclear ribosomal cluster (ITS) and intron 2 of the glyceraldehyde-3-phosphate dehydrogenase gene (G3PD) were sequenced for isolates from PFD-affected sweet orange and KLA-affected Key Limes collected in the United States (Florida), Brazil (Sao Paulo), Mexico, Belize, Costa Rica, and the Dominican Republic to determine if there are consistent genetic differences between PFD and KLA isolates over the geographic area where these diseases occur. Based on the sequence data, isolates clustered into two well-supported clades with little or no sequence variation among isolates within clades. One clade (PFD clade) contained PFD isolates from all countries sampled plus a few isolates from flowers of Key Lime in Brazil. The other clade (KLA clade) contained KLA isolates from Key Lime foliage from all countries sampled and one isolate from flowers of sweet orange in Mexico. In greenhouse inoculations with PFD and KLA isolates from Florida, isolates from both clades produced PFD symptoms on Orlando tangelo flowers, but KLA-clade isolates produced significantly less severe symptoms. PFD-clade isolates were not pathogenic to Key Lime foliage, confirming previous studies. The differentiation of PFD and KLA isolates into two well-supported clades and the pathogenicity data indicate that PFD and KLA are caused by distinct phylogenetic lineages of C. acutatum that are also biologically distinct. PFD is a recently described disease (first reported in 1979) relative to KLA (first reported in 1912) and it had been proposed that strains causing PFD evolved from strains causing KLA eventually losing pathogenicity to Key Lime foliage. We reject the hypothesis that PFD strains have diverged from KLA strains recently based on estimated divergence times of haplotypes and it appears that PFD and KLA strains have been dispersed throughout the Americas independently in association with each host.
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indole derivatives produced by the fungus colletotrichum acutatum causing Lime anthracnose and postbloom fruit drop of citrus
Fems Microbiology Letters, 2003Co-Authors: Kuang-ren Chung, L W Timmer, Turksen Shilts, Umran Erturk, P P UengAbstract:Postbloom fruit drop (PFD) of citrus and Key Lime anthracnose (KLA) are caused by Colletotrichum acutatum. Both fungal isolates can infect flower petals, induce young fruit abscission and result in severe yield loss on many citrus cultivars. Previous studies revealed that infection of citrus flowers by C. acutatum caused higher levels of indole-3-acetic acid (IAA), which could be synthesized from the host plant and/or the fungal pathogen. The ability for IAA production by C. acutatum isolates was investigated. Similar to many microorganisms, the production of indole compounds in the medium by C. acutatum was dependent solely on the presence of tryptophan (Trp). In total, 14 PFD and KLA fungal isolates were tested, and revealed that they all were capable of utilizing Trp as a precursor to synthesize IAA and other indole derivatives. High-performance liquid chromatography analysis and chromogenic stains after a fluorescence thin-layer chromatography separation unambiguously identified IAA, tryptophol (TOL), indole-acetaldehyde, indole-acetamide (IAM), indole-pyruvic acid, and indole-lactic acid (ILA) from cultures supplemented with Trp. The data suggest that C. acutatum may synthesize IAA using various pathways. Interestingly, increasing Trp concentrations drastically increased the levels of TOL and ILA, but not IAA and IAM. The ability of C. acutatum to produce IAA and related indole compounds may in part contribute to the increased IAA levels in citrus flowers after infection.
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engineering a genetic transformation system for colletotrichum acutatum the causal fungus of Lime anthracnose and postbloom fruit drop of citrus
Fems Microbiology Letters, 2002Co-Authors: Kuang-ren Chung, Turksen Shilts, Wei Li, L W TimmerAbstract:Postbloom fruit drop (PFD) of citrus is caused by Colletotrichum acutatum. PFD isolates infect flower petals, induce abscission of small fruit and can cause severe yield loss on most citrus cultivars. Isolates from Key Lime anthracnose (KLA) cause that disease on the Mexican Lime, but also cause PFD on sweet orange. Both PFD and KLA isolates exhibited resistance to the common selection agents including hygromycin, bialaphos, benomyl and geneticin/G418. A genetic transformation system was developed for C. acutatum to confer resistance to sulfonylurea (chlorimuron ethyl) by expressing an acetolactate synthase gene (sur) cassette from Magnaporthe grisea. The protocol was tested on 11 different KLA and PFD isolates. The transformation frequencies were highly variable among isolates and among experiments (0–17.9 per μg circular DNA using 107 protoplasts). Southern blot analysis of transformants indicated that the plasmid vector was randomly integrated in multiple copies into the genome of C. acutatum. Addition of restriction enzymes or use of a vector with homologous sequences did not change the transformation frequencies, but tended to reduce the number integrated. Over 97% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. Of 300 transformants tested, three were unable to cause necrotic lesions on detached Key Lime leaves. The transformation method opens up opportunities for the genetic manipulation of C. acutatum.
Giovanni Dugo - One of the best experts on this subject based on the ideXlab platform.
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multidimensional enantio gas chromtography mass spectrometry and gas chromatography combustion isotopic ratio mass spectrometry for the authenticity assessment of Lime essential oils c aurantifolia swingle and c latifolia tanaka
Journal of Chromatography A, 2012Co-Authors: Ivana Bonaccorsi, Paola Dugo, Luigi Mondello, Danilo Sciarrone, Luisa Schipilliti, Giovanni DugoAbstract:This article focuses on the genuineness assessment of Lime oils (Citrus aurantifolia Swingle and C. latifolia Tanaka), by Multi Dimensional Gas Chromatography (MDGC) to determine the enantiomeric distribution of α-thujene, camphene, β-pinene, sabinene, α-phellandrene, β-phellandrene, limonene, linalool, terpinen-4-ol, α-terpineol and by gas chromatography-combustion isotope ratio mass spectrometry (GC-C-IRMS) to determine the isotopic ratios of α-pinene, β-pinene, limonene, α-terpineol, neral, geranial, β-caryophyllene, trans-α-bergamotene, germacrene B. To the author's knowledge this is the first attempt to assess the authenticity and differentiate Persian Lime from Key Lime oils by GC-C-IRMS. The results of the two analytical approaches were compared. The simultaneous use of the two techniques provides more reliable capability to detect adulteration in Citrus essential oils. In fact, in some circumstance only one of the two techniques allows to discriminate adulterated or contaminated oils. In cases where only small anomalies are detected by the two techniques due to subtle adulterations, their synergic use allows to express judgments. The advantage of both techniques is the low number of components the analyst must evaluate, reducing the complexity of the data necessary to deal with. Moreover, the conventional analytical approach based on the evaluation of the whole volatile fraction can fail to reveal the quality of the oils, if the adulteration is extremely subtle.
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analytical characterization of industrial essential oils from fruits and leaves of c aurantifolia tan and c latifolia swing
Journal of Essential Oil Research, 2011Co-Authors: Ivana Bonaccorsi, Paola Dugo, Luigi Mondello, Giovanni Dugo, Danilo Sciarrone, Luis HaroguzmanAbstract:Abstract The physicochemical indices, the composition of the volatile fraction, the enantiomeric ratios of some volatile components and the oxygen heterocyclic fraction of cold-pressed Key Lime oils (types A and B), Persian Lime oils, and petitgrain Lime oils are reported. The volatile fraction of cold-pressed Persian Lime oil is characterized by a higher content of limonene, γ-terpinene and esters and a lower content of β-pinene, sesquiterpene hydrocarbons, alcohols and aldehydes than cold-pressed Key Lime oils. In petitgrain oils the oxygenated compounds are present at levels higher than the peel oils. Oxypeucedanin, probably due to the extraction technology, was almost absent in cold-pressed Key Lime type A, while it is present in cold-pressed Key Lime type B and in Persian Lime oil. The en-separation was performed by direct enantioselective GC (esGC) and by multidimensional GC (MDGC) to obtain the most appropriate antiomeric separation of all the components analyzed. The enantiomeric excess of S-(-)-α...
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characterization of cold pressed Key and persian Lime oils by gas chromatography gas chromatography mass spectroscopy high performance liquid chromatography and physicochemical indices
Journal of Agricultural and Food Chemistry, 1997Co-Authors: Paola Dugo, Luigi Mondello, G. Lamonica, Giovanni DugoAbstract:The physicochemical indices and the qualitative and quantitative composition of the volatile fraction and the oxygenated heterocyclic fraction of cold-pressed Key Lime oil (types A and B) and Persian Lime oil are reported. The volatile fraction of Persian Lime oil is characterized by a higher content of limonene, γ-terpinene, esters, and monoterpene aldehydes and a lower content of β-pinene + sabinene, sesquiterpenes, and aliphatic aldehydes than Key Lime oils. Oxypeucedanin was not detected in Key Lime oil type A, while it is present in Key Lime oil type B and Persian Lime oil. This is probably due to the extraction technology used for Key Lime oil type A, which allows the essential oil to come into contact with the juice. Under these conditions, the epoxy ring of oxypeucedanin is opened by hydrolysis to form oxypeucedanin hydrate. Keywords: Citrus aurantifolia Swingle; cold-pressed Key Lime oil; type A; type B; Citrus latifolia Tanaka; cold-pressed Persian Lime oil; volatile fraction; limonene; γ-terpin...
Kuang-ren Chung - One of the best experts on this subject based on the ideXlab platform.
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the colletotrichum acutatum gene encoding a putative ph responsive transcription regulator is a Key virulence determinant during fungal pathogenesis on citrus
Molecular Plant-microbe Interactions, 2007Co-Authors: Bangjau You, Mathias Choquer, Kuang-ren ChungAbstract:Postbloom fruit drop of citrus and Key Lime anthracnose (KLA) are caused by different pathotypes of Colletotrichum acutatum. Both pathotypes are pathogenic to citrus flowers, resulting in blossom blight and induction of young fruit abscission. Two fungal mutants defective in pathogenicity were recovered from a KLA pathotype after Agrobacterium-mediated mutagenesis. A PacCKLAP2 gene encoding a polypeptide that resembles many pH-responsive PacC/Rim101 transcription regulators in fungi was identified from one of the mutants, and functionally characterized to play a crucial role in pathogenesis to both Key Lime leaves and citrus flowers. Gene disruption at the PacCKLAP2 locus created fungal mutants that were hypersensitive to alkaline pH, altered in conidium and appressorium production and germination, and concomitant with reduced virulence to both tissues. The pacCklap2 null mutants had lower alkaline phosphatase and protease activities, but increased pectolytic and lipolytic activities. The mutants initiate...
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indole derivatives produced by the fungus colletotrichum acutatum causing Lime anthracnose and postbloom fruit drop of citrus
Fems Microbiology Letters, 2003Co-Authors: Kuang-ren Chung, L W Timmer, Turksen Shilts, Umran Erturk, P P UengAbstract:Postbloom fruit drop (PFD) of citrus and Key Lime anthracnose (KLA) are caused by Colletotrichum acutatum. Both fungal isolates can infect flower petals, induce young fruit abscission and result in severe yield loss on many citrus cultivars. Previous studies revealed that infection of citrus flowers by C. acutatum caused higher levels of indole-3-acetic acid (IAA), which could be synthesized from the host plant and/or the fungal pathogen. The ability for IAA production by C. acutatum isolates was investigated. Similar to many microorganisms, the production of indole compounds in the medium by C. acutatum was dependent solely on the presence of tryptophan (Trp). In total, 14 PFD and KLA fungal isolates were tested, and revealed that they all were capable of utilizing Trp as a precursor to synthesize IAA and other indole derivatives. High-performance liquid chromatography analysis and chromogenic stains after a fluorescence thin-layer chromatography separation unambiguously identified IAA, tryptophol (TOL), indole-acetaldehyde, indole-acetamide (IAM), indole-pyruvic acid, and indole-lactic acid (ILA) from cultures supplemented with Trp. The data suggest that C. acutatum may synthesize IAA using various pathways. Interestingly, increasing Trp concentrations drastically increased the levels of TOL and ILA, but not IAA and IAM. The ability of C. acutatum to produce IAA and related indole compounds may in part contribute to the increased IAA levels in citrus flowers after infection.
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engineering a genetic transformation system for colletotrichum acutatum the causal fungus of Lime anthracnose and postbloom fruit drop of citrus
Fems Microbiology Letters, 2002Co-Authors: Kuang-ren Chung, Turksen Shilts, Wei Li, L W TimmerAbstract:Postbloom fruit drop (PFD) of citrus is caused by Colletotrichum acutatum. PFD isolates infect flower petals, induce abscission of small fruit and can cause severe yield loss on most citrus cultivars. Isolates from Key Lime anthracnose (KLA) cause that disease on the Mexican Lime, but also cause PFD on sweet orange. Both PFD and KLA isolates exhibited resistance to the common selection agents including hygromycin, bialaphos, benomyl and geneticin/G418. A genetic transformation system was developed for C. acutatum to confer resistance to sulfonylurea (chlorimuron ethyl) by expressing an acetolactate synthase gene (sur) cassette from Magnaporthe grisea. The protocol was tested on 11 different KLA and PFD isolates. The transformation frequencies were highly variable among isolates and among experiments (0–17.9 per μg circular DNA using 107 protoplasts). Southern blot analysis of transformants indicated that the plasmid vector was randomly integrated in multiple copies into the genome of C. acutatum. Addition of restriction enzymes or use of a vector with homologous sequences did not change the transformation frequencies, but tended to reduce the number integrated. Over 97% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. Of 300 transformants tested, three were unable to cause necrotic lesions on detached Key Lime leaves. The transformation method opens up opportunities for the genetic manipulation of C. acutatum.
Ahmed Dalel - One of the best experts on this subject based on the ideXlab platform.
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Preferential Disomic Segregation and C. micrantha/C. medica Interspecific Recombination in Tetraploid 'Giant Key' Lime; Outlook for Triploid Lime Breeding
'Frontiers Media SA', 2020Co-Authors: Ahmed Dalel, Froelicher Yann, Curk Franck, Evrard Jean, Ollitrault PatrickAbstract:International audienceThe triploid 'Tahiti' Lime (C. x latifolia(Yu. Tanaka) Tanaka) naturally originated from a merger between a haploid ovule of lemon (C. x limon(L.) Burm) and a diploid pollen from a 'Mexican' Lime (C. x aurantiifolia(Christm.) Swing). The very limited natural inter-varietal diversity and gametic sterility ofC. latifoliarequires a phylogenomic based reconstruction breeding strategy to insure its diversification. We developed a strategy based on interploid hybridization between diploid lemon and the doubled diploid 'Giant Key' Lime. This Lime is a doubled diploid of 'Mexican' Lime, itself a natural interspecific F1 hybrid betweenC. medicaL. andC. micranthaWester. For an optimized breeding program, we analyzed the meiotic behavior of the allotetraploid Lime, the genetic structure of its diploid gametes, the interspecific recombination betweenC. medicaandC. micrantha, and constructed its genetic map. A population of 272 triploid hybrids was generated using 'Giant Key' Lime as pollinator. One hundred fifty-eight SNPs diagnostic ofC. micrantha,regularly distributed throughout the citrus genome were successfully developed and applied. The genetic structure of the diploid gametes was examined based onC. micranthadoses along the genome. The diploid gametes transmitted in average 91.17% of the parental interspecificC. medica/C. micranthaheterozygosity. Three chromosomes (2, 8, and 9) showed disomic segregation with high preferential pairing values, while the remaining chromosomes showed an intermediate inheritance with a preferential disomic trend. A total of 131 SNPs were assigned to nine linkage groups to construct the genetic map. It spanned 272.8 cM with a low average recombination rate (0.99 cM Mb(-1)) and high synteny and colinearity with the reference clementine genome. Our results confirmed that an efficient reconstruction breeding strategy for 'Tahiti' Lime is possible, based on interploid hybridization using a doubled diploid ofC. aurantiifolia. The tetraploid parent should be selected for favorable agronomic traits and its genetic value should be efficiently inherited by the progeny thanks to transmission of the high level of parental heterozygosity. However, it would require developing numerous progeny to overcome the linkage drag caused by the limited interspecific recombination associated with the predominant disomic inheritance
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Preferential disomic segregation and C. micrantha/C. medica interspecific recombination in tetraploid 'Giant Key' Lime; outlook for triploid Lime breeding
'Frontiers Media SA', 2020Co-Authors: Ahmed Dalel, Froelicher Yann, Curk Franck, Evrard Jean-charles, Ollitrault PatrickAbstract:The triploid 'Tahiti' Lime (C. x latifolia (Yu. Tanaka) Tanaka) naturally originated from a merger between a haploid ovule of lemon (C. x limon (L.) Burm) and a diploid pollen from a 'Mexican' Lime (C. x aurantiifolia (Christm.) Swing). The very limited natural inter-varietal diversity and gametic sterility of C. latifolia requires a phylogenomic based reconstruction breeding strategy to insure its diversification. We developed a strategy based on interploid hybridization between diploid lemon and the doubled diploid 'Giant Key' Lime. This Lime is a doubled diploid of 'Mexican' Lime, itself a natural interspecific F1 hybrid between C. medica L. and C. micrantha Wester. For an optimized breeding program, we analyzed the meiotic behavior of the allotetraploid Lime, the genetic structure of its diploid gametes, the interspecific recombination between C. medica and C. micrantha, and constructed its genetic map. A population of 272 triploid hybrids was generated using 'Giant Key' Lime as pollinator. One hundred fifty-eight SNPs diagnostic of C. micrantha, regularly distributed throughout the citrus genome were successfully developed and applied. The genetic structure of the diploid gametes was examined based on C. micrantha doses along the genome. The diploid gametes transmitted in average 91.17% of the parental interspecific C. medica/C. micrantha heterozygosity. Three chromosomes (2, 8, and 9) showed disomic segregation with high preferential pairing values, while the remaining chromosomes showed an intermediate inheritance with a preferential disomic trend. A total of 131 SNPs were assigned to nine linkage groups to construct the genetic map. It spanned 272.8 cM with a low average recombination rate (0.99 cM Mb-1) and high synteny and colinearity with the reference clementine genome. Our results confirmed that an efficient reconstruction breeding strategy for 'Tahiti' Lime is possible, based on interploid hybridization using a doubled diploid of C. aurantiifolia. The tetraploid parent should be selected for favorable agronomic traits and its genetic value should be efficiently inherited by the progeny thanks to transmission of the high level of parental heterozygosity. However, it would require developing numerous progeny to overcome the linkage drag caused by the limited interspecific recombination associated with the predominant disomic inheritance
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Analysis of the inheritance of chromosomal fragments and the phenotype-genotype association in populations of triploid citrus fruits deriving from an interspecific origin.
2020Co-Authors: Ahmed DalelAbstract:De nombreux programmes d’amélioration variétale des agrumes basés sur la manipulation de la ploïdie ont été développés pour répondre aux attentes du marché et des agriculteurs. L’absence de pépins est un caractère très recherché pouvant être obtenu par la triploïdisation. Des hybrides triploïdes peuvent résulter de la formation des gamètes non-réduits dans les croisements 2x X 2x ou être créés par hybridations interploïdes (2x X 4x ou 4x X 2x). Les agrumes se caractérisent par des structures phylogénomiques complexes très hétérozygotes souvent d’origine interspécifique. La compréhension des mécanismes de la formation des 2n gamètes et la connaissance des structures phylogénomiques sont essentielles pour optimiser les stratégies d’amélioration. C’est dans ce cadre qu’ont été fixés les objectifs de cette thèse : (i) mettre en place une approche basée sur le GBS pour décrypter les structures phylogénomiques des agrumes de différentes ploïdies, (ii) évaluer le potentiel d’une variété diploïde-doublée de Limettier pour la mise en place d’une stratégie de reconstruction d’un idéotype triploïde ‘Tahiti’ à partir d’hybridations interploïdes, (iii) étudier les variations phénotypiques au sein de populations de mandariniers 2x et 3x en fonction de la ploïdie et de la variété utilisée comme parent femelle, et (iv) établir les cartes génétiques des géniteurs des populations de mandariniers hybrides et analyser la structure des gamètes non-réduits, en utilisant le GBS.Les données GBS d’un panel représentatif des quatre taxons ancestraux des agrumes cultivés ont révélé 15946 marqueurs SNP diagnostiques (DSNP) des taxons de base. Ces marqueurs servent de référentiel pour inférer les structures phylogénomiques des agrumes selon une méthode basée sur le maximum de vraisemblance. Cette méthode a été implémentée en ligne sous le nom de « TraceAncestor » et permet de générer les caryotypes d’espèces dont les génomes sont sous forme de mosaïques de grands fragments issus de plusieurs taxons de base. Cet outil a permis de décrypter, pour la première fois, les génomes de 16 variétés diploïdes, quatre variétés triploïdes et une variété tétraploïde d’agrumes.158 marqueurs KASPar diagnostiques de C. micrantha ont été développés pour suivre la ségrégation de la Lime 4x ‘Giant Key’. Le génotypage de 272 hybrides 3x de Limettiers issus d’un croisement entre divers citronniers 2x et le Limettier 4x ‘Giant Key’ a révélé une ségrégation principalement disomique et des taux de transmission de l’hétérozygotie parentale élevés. Bien que limitées, les recombinaisons interspécifiques ont permis d’établir la première carte génétique de ‘Giant Key’ qui témoigne d’une synténie et colinéarité fortes avec la séquence de référence du clémentinier. Nos résultats appuient l’hypothèse d’une origine interploïde de la Lime 3x ‘Tahiti’ et valident la stratégie de sa diversification par reconstruction phylogénomique.Des populations de mandariniers hybrides 2x et 3x issues de croisements réciproques entre la mandarine ‘Fortune’ et le tangor ‘Ellendale’ ont été phénotypées et génotypées par GBS. L'analyse des données phénotypiques a révélé un effet du dosage parental sur les taux de sucre et d’acidité. Deux cartes génétiques parentales saturées ont été développées avec les données GBS des populations diploïdes. Le mécanisme SDR a été identifié à l’origine des diplogamètes femelles ayant produit les hybrides triploïdes, et des taux de recombinaison se sont révélés plus élevés chez les 2n gamètes que chez les gamètes haploïdes. Deux QTLs pour le poids du jus et l’acidité ont été détectés chez les triploïdes. L’analyse des distorsions de ségrégation a révélé, au début du chromosome 7, une région porteuse d’un gène impliqué dans l’auto-incompatibilité gamétophytique.Les connaissances, les outils et les méthodes développés dans le cadre de cette thèse permettront une exploitation plus efficace de la diversité des agrumes dans les programmes d’innovation variétale des triploïdes.Many citrus breeding programs based on ploidy manipulation have been developed worldwide to meet market and farmers’ expectations. Seedlessness is a particularly sought-after characteristic that can be obtained by triploidization. Triploid hybrids can be recovered from the formation of unreduced gametes through 2x X 2x crosses, or through interploid hybridizations (2x X 4x or 4x X 2x). In addition, citrus fruits are characterized by highly heterozygous and complex phylogenomic structures, often deriving from an interspecific origin. Understanding the mechanisms underlying the formation of diploid gametes and discovering the phylogenomic structures are essential for optimizing citrus breeding schemes. Within this framework, the objectives of this thesis have been: (i) the implementation of a genotyping by sequencing (GBS) based approach for deciphering the phylogenomic structures of citrus fruits with different levels of ploidy, (ii) the evaluation of the potential of a doubled-diploid Lime variety for implementing a reconstruction breeding strategy of a triploid ideotype based on interploid hybridization, (iii) the study of the phenotypic variations within diploid and triploid mandarin hybrid populations according to the ploidy level and the variety used as female parent, and (iv) the establishment of genetic maps of the parents of mandarin hybrid populations and the analysis of unreduced gamete structures from GBS data.The analysis of the GBS data of varieties belonging to the Citrus genus, some of which representing the four ancestral taxa, revealed 15946 SNP markers diagnostic (DSNP) of the Citrus basic taxa. These markers were used to infer the phylogenomic structures of citrus species and varieties, based on a maximum likelihood method. The pipeline has been implemented online under the name of “TraceAncestor”. It allows generating the phylogenomic karyotypes of species whose reproductive behavior resulted in an interspecific mosaic of large genomic fragments from several ancestral taxa. This tool was successfully used to decipher, for the first time, the karyotypes of 16 diploid citrus varieties, four triploid and one tetraploid Lime.One hundred and fifty eight KASPar markers diagnostic of C. micrantha have been successfully developed to analyse the segregation of the doubled-diploid 'Giant Key' Lime. GBS data of 272 triploid hybrid Limes, coming from a cross between various diploid lemons and the tetraploid 'Giant Key' Lime, revealed a predominantly disomic segregation and a high parental heterozygosity restitution. Although limited, the interspecific recombination allowed to establish the first genetic map of the tetraploid 'Giant Key' Lime, which showed high synteny and colinearity with the clementine reference genome. Our results reinforce the hypothesized interploid hybridization origin of the triploid ‘Tahiti’ Lime, and validate the ‘Tahiti’ ideotype diversification project based on a phylogenomic reconstructing strategy.Diploid and triploid mandarin hybrid populations resulting from reciprocal crosses between ‘Fortune’ mandarin and ‘Ellendale’ tangor have been phenotyped and genotyped by GBS. Statistical analyses of phenotypic data highlighted a parental dosage effect which affected sugar and acidity levels. Two high-density parental genetic maps were developed using GBS data of the diploid populations. The SDR mechanism was identified at the origin of the female diplogametes that generated the triploid hybrids. Higher recombination rates were found in the 2n gametes than in the haploid ones. Two QTLs for juice weight and acidity level were detected in triploids. The analysis of segregation distortions evidenced, at the beginning of chromosome 7, a probable major pollen gene for gametophytic self-incompatibility.Acquired knowledge as well as the tools and methods developed during this thesis will allow a more efficient exploitation of citrus diversity for implementing triploid breeding programs
Jude W Grosser - One of the best experts on this subject based on the ideXlab platform.
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immature embryo rescue culture and seedling development of acid citrus fruit derived from interploid hybridization
Plant Cell Tissue and Organ Culture, 2005Co-Authors: Z Viloria, Jude W Grosser, B BrachoAbstract:Interploid sexual hybridizations were completed in 2001 and 2002 between seven lemon (Citrus limon(L.) Burm. f.) varieties, ‘Key’ Lime (C. aurantifolia (Cristm.) Swing), Palestine sweet Lime (C. imettioides Tan.), ‘Lakeland’ Limequat (C. aurantifolia x Fortunella japonica (Thumb.) Swing.), and ‘Etrog’ citron (C. medica L.) as diploid progenitors and four allotetraploid somatic hybrids (‘Key’ Lime + ‘Valencia’ orange, ‘Hamlin’ orange + ‘Femminello’ lemon, ‘Valencia’ orange + Rough lemon, and ‘Valencia’ orange+ ‘Femminello’ lemon) in efforts to generate improved seedless triploid acid fruit hybrids. Efficient recovery of triploid progeny from such crosses requires embryo rescue to avoid embryo abortion due to endosperm failure. Germination of rescued genetically diverse immature embryos was induced on two culture media (EME and Gamborg’s B5), with two sucrose concentrations (50 or 70 g l−1). All media contained 0.5 g l−1 malt extract and 4.50 μM GA3. Germination of globular, heart and torpedo shaped embryos (defined as small embryos) was significantly (p < 0.05) affected by medium and genotype. Gamborg’s medium induced 82.89% germination. Of germinated embryos, 11–65% developed into normal plants with differences among crosses. Cotyledonary embryos (defined as immature embryos with fully developed cotyledons) germinated and developed into normal plants at higher rates than less-developed embryos. In efforts to improve the efficiency of plant recovery, small embryos from ‘Todo el ano’ × HF and ‘Lisbon’ × HF crosses conducted during 2002 were rescued and cultured on three media (MS, Gamborg’s, and RMA) for comparison. Media did not significantly affect the proportion of normal plant recovery.
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screening triploid hybrids of lakeland Limequat for resistance to citrus canker
Plant Disease, 2004Co-Authors: Z Viloria, D L Drouillard, J H Graham, Jude W GrosserAbstract:ABSTRACT Resistance of citrus genotypes to Xanthomonas axonopodis pv. citri, the cause of Asiatic citrus canker (ACC), was evaluated by injection infiltration of 103 and 104 CFU/ml through stomates on the abaxial surface of immature leaves. Citrus genotypes for screening comprised two autotetraploids and nine triploid hybrids of ‘Lakeland’ Limequat (Citrus aurantifolia × Fortunella japonica) and their progenitors (‘Lakeland’ Limequat, the autotetraploids ‘Femminello’ lemon (Citrus limon) and ‘Giant Key’ Lime (C. aurantifolia), and the somatic hybrids ‘Key’ [also known as ‘Mexican’] Lime + ‘Valencia’ orange and ‘Hamlin’ orange + ‘Femminello’ lemon). ‘Meiwa’ kumquat (Fortunella crassifolia) and ‘Pineapple’ sweet orange (C. sinensis) were used as known resistant and susceptible standards, respectively. Lesion number per inoculation site and bacterial population per lesion were recorded 15 to 19 days after inoculation. The assay was performed four times during a spring-summer-fall period under greenhouse cond...
