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A Conserved Ethylene Biosynthesis Enzyme Leads to Andromonoecy in Two Cucumis Species
Adnane Boualem, Christelle Troadec, Irina Kovalski, Marie-Agnes Sari, Rafael Perl-Treves, Abdelhafid Bendahmane
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0006144
Abstract: Andromonoecy is a widespread sexual system in angiosperms, characterized by plants carrying both male and bisexual flowers. Monoecy is characterized by the presence of both male and female flowers on the same plant. In cucumber, these sexual forms are controlled by the identity of the alleles at the M locus. In melon, we recently showed that the transition from monoecy to andromonoecy result from a mutation in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, CmACS-7. To isolate the andromonoecy gene in cucumber we used a candidate gene approach in combination with genetical and biochemical analysis. We demonstrated co-segregation of CsACS2, a close homolog of CmACS-7, with the M locus. Sequence analysis of CsACS2 in cucumber accessions identified four CsACS2 isoforms, three in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed the four isoforms in Escherichia coli and assayed their activity in vitro. Like in melon, the isoforms from the andromonoecious lines showed reduced to no enzymatic activity and the isoform from the monoecious line was active. Consistent with this, the mutations leading andromonoecy were clustered in the active site of the enzyme. Based on this, we concluded that active CsACS2 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. Consistent with this, CsACS2, like CmACS-7 in melon, is expressed specifically in carpel primordia of buds determined to develop carpels. Following ACS expression, inter-organ communication is likely responsible for the inhibition of stamina development. In both melon and cucumber, flower unisexuality seems to be the ancestral situation, as the majority of Cucumis species are monoecious. Thus, the ancestor gene of CmACS-7/CsACS2 likely have controlled the stamen development before speciation of Cucumis sativus (cucumber) and Cucumis melo (melon) that have diverged over 40 My ago. The isolation of the genes for andromonoecy in Cucumis species provides a molecular basis for understanding how sexual systems arise and are maintained within and between species.
Phenotypic and fine genetic characterization of the D locus controlling fruit acidity in peach
Karima Boudehri, Abdelhafid Bendahmane, Ga?lle Cardinet, Christelle Troadec, Annick Moing, Elisabeth Dirlewanger
BMC Plant Biology , 2009, DOI: 10.1186/1471-2229-9-59
Abstract: In order to generate a high-resolution linkage map in the vicinity of the D locus, 1,024 AFLP primer combinations were screened using DNA of bulked acid and low-acid segregants. We also screened a segregating population of 1,718 individuals for chromosomal recombination events linked to the D locus and identified 308 individuals with recombination events close to D. Using these recombinant individuals we delimited the D locus to a genetic interval of 0.4 cM. We also constructed a peach BAC library of 52,000 clones with a mean insert size of 90 kb. The screening of the BAC library with markers tightly linked to D locus indicated that 1 cM corresponds to 250 kb at the vicinity of the D locus.In the present work we presented the first high-resolution genetic map of D locus in peach. We also constructed a peach BAC library of approximately 15× genome equivalent. This fine genetic and physical characterization of the D locus is the first step towards the isolation of the gene(s) underlying fruit acidity in peach.Peach [Prunus persica (L.) Batsch] belongs to the Spiraeoideae subfamily of the Rosaceae [1]. The Prunus genus is characterized by species producing drupes as fruit, and contains a significant number of economically important fruit tree species such as almond (Prunus dulcis (Mill.)), apricot (Prunus armeniaca L.), sweet cherry (Prunus avium L.), sour cherry (Prunus cerasus L.) and plum (Prunus domestica L.).Compared to other tree species, peach has a relative small diploid genome (290 Mb) [2], and a short juvenile phase (two to three years). Therefore, peach is considered as a model species for Rosaceae family and a physical map of its genome has been initiated [3].Among fruit producing rosaceous crops, peach is the second most important fruit crop in Europe after apple and the third worldwide (FAOSTAT: http://faostat.fao.org/ webcite). However, the consumption of peaches and nectarines is stagnant due to the low quality of fruits that are harvested at an immatur
An Induced Mutation in Tomato eIF4E Leads to Immunity to Two Potyviruses
Florence Piron,Maryse Nicola?,Silvia Mino?a,Elodie Piednoir,André Moretti,Aurélie Salgues,Dani Zamir,Carole Caranta,Abdelhafid Bendahmane
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0011313
Abstract: The characterization of natural recessive resistance genes and Arabidopsis virus-resistant mutants have implicated translation initiation factors of the eIF4E and eIF4G families as susceptibility factors required for virus infection and resistance function.
A new mutant genetic resource for tomato crop improvement by TILLING technology
Silvia Minoia, Angelo Petrozza, Olimpia D'Onofrio, Florence Piron, Giuseppina Mosca, Giovanni Sozio, Francesco Cellini, Abdelhafid Bendahmane, Filomena Carriero
BMC Research Notes , 2010, DOI: 10.1186/1756-0500-3-69
Abstract: To apply TILLING to tomato, a new mutant collection was generated in the genetic background of the processing tomato cultivar Red Setter by treating seeds with two different ethylemethane sulfonate doses (0.7% and 1%). An associated phenotype database, LycoTILL, was developed and a TILLING platform was also established. The interactive and evolving database is available online to the community for phenotypic alteration inquiries. To validate the Red Setter TILLING platform, induced point mutations were searched in 7 tomato genes with the mismatch-specific ENDO1 nuclease. In total 9.5 kb of tomato genome were screened and 66 nucleotide substitutions were identified. The overall mutation density was estimated and it resulted to be 1/322 kb and 1/574 kb for the 1% EMS and 0.7% EMS treatment respectively.The mutation density estimated in our collection and its comparison with other TILLING populations demonstrate that the Red Setter genetic resource is suitable for use in high-throughput mutation discovery. The Red Setter TILLING platform is open to the research community and is publicly available via web for requesting mutation screening services.Tomato (Solanum lycopersicum) is one of the most important vegetable plants in the world. Its fruits are end products both for the fresh market and food processing industry. Tomato presents a relatively small genome highly syntenic to others economically important Solanaceae species and was selected as a reference species for sequencing a Solanaceae genome. In addition to the availability of a number of genomic resources, including transcriptome [1-3] and metabolome [4], large collections of genetic resources are available to dissect the biochemical and the metabolic pathways in tomato [5]. Large EMS and fast neutron mutant collections, in the background of M82 tomato cultivar, have been generated and more then 3,000 phenotype alterations catalogued [6]. An EMS-induced mutation library of the miniature dwarf tomato cultivar Mi
Engineering Melon Plants with Improved Fruit Shelf Life Using the TILLING Approach
Fatima Dahmani-Mardas,Christelle Troadec,Adnane Boualem,Sylvie Lévêque,Abdullah A. Alsadon,Abdullah A. Aldoss,Catherine Dogimont,Abdelhafid Bendahmane
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015776
Abstract: Fruit ripening and softening are key traits that have an effect on food supply, fruit nutritional value and consequently, human health. Since ethylene induces ripening of climacteric fruit, it is one of the main targets to control fruit over ripening that leads to fruit softening and deterioration. The characterization of the ethylene pathway in Arabidopsis and tomato identified key genes that control fruit ripening.
UTILLdb, a Pisum sativum in silico forward and reverse genetics tool
Marion Dalmais, Julien Schmidt, Christine Le Signor, Francoise Moussy, Judith Burstin, Vincent Savois, Gregoire Aubert, Veronique Brunaud, Yannick de Oliveira, Cecile Guichard, Richard Thompson, Abdelhafid Bendahmane
Genome Biology , 2008, DOI: 10.1186/gb-2008-9-2-r43
Abstract: Mutational approaches have been widely exploited in breeding and basic research. In the genomic era, the completion of the sequencing of several plant genomes has enabled the development of reverse genetics strategies, where one first identifies a target gene based on the functional annotation of its sequence, and then proceeds with the phenotypic characterization of mutant alleles. Several mutagenesis techniques are dedicated to this approach, notably RNA interference suppression [1,2] and insertional mutagenesis by transposon tagging [3,4] or Agrobacterium T-DNA insertion [5]. These methods, however, are still mainly based on Agrobacterium T-DNA vectors and, thus, rely on the ability of a given plant species to be transformed. On the other hand, chemical mutagenesis based on an alkylating agents like ethylmethane sulfonate (EMS) [6] provides an easy and cost-effective way to saturate a genome with mutations. TILLING (targeting induced local lesions in genomes) uses EMS mutagenesis coupled with gene-specific detection of single-nucleotide mutations [7-9]. This reverse genetic strategy encompasses all types of organisms [10-14] and can be automated in a high throughput mode, which is an absolute necessity to match the speed of candidate gene discovery.The success of the TILLING approach relies on the construction of high quality mutant libraries. Ideally, the mutant population is phenotyped so that in silico analysis of the mutant lines can be carried out. To date, phenotypic databases can be found for tomato [15], rice [16], Lotus japonicus [13] and Arabidopsis [17], and a searchable collection of phenotypic mutants is available for Zea mays [18], Pisum sativum [19] and Arabidopsis thaliana [20].Pea (P. sativum) belongs to the Leguminoseae family, which provides excellent dietary components with health-promoting benefits and offers the important ecological advantage of contributing to the development of low input farming systems by fixing atmospheric nitrogen and f
Towards a TILLING platform for functional genomics in Piel de Sapo melons
Mireia González, Meihong Xu, Cristina Esteras, Cristina Roig, Antonio J Monforte, Christelle Troadec, Marta Pujol, Fernando Nuez, Abdelhafid Bendahmane, Jordi Garcia-Mas, Belén Picó
BMC Research Notes , 2011, DOI: 10.1186/1756-0500-4-289
Abstract: A new ethyl methanesulfonate-mutagenized (EMS) melon population was generated for the first time in an andromonoecious non-climacteric inodorus Piel de Sapo genetic background. Diverse mutant phenotypes in seedlings, vines and fruits were observed, some of which were of possible commercial interest. The population was first screened for mutations in three target genes involved in disease resistance and fruit quality (Cm-PDS, Cm-eIF4E and Cm-eIFI(iso)4E). The same genes were also tilled in the available monoecious and climacteric cantalupensis EMS melon population. The overall mutation density in this first Piel de Sapo TILLING platform was estimated to be 1 mutation/1.5 Mb by screening four additional genes (Cm-ACO1, Cm-NOR, Cm-DET1 and Cm-DHS). Thirty-three point mutations were found for the seven gene targets, six of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was demonstrated for a loss-of-function mutation in the Phytoene desaturase gene, which is involved in carotenoid biosynthesis.The TILLING approach was successful at providing new mutations in the genetic background of Piel de Sapo in most of the analyzed genes, even in genes for which natural variation is extremely low. This new resource will facilitate reverse genetics studies in non-climacteric melons, contributing materially to future genomic and breeding studies.Melon (Cucumis melo L.) is an important vegetable crop. Genetic and genomic information for this crop is increasing significantly due to several national and international projects [1]. A broad range of genomic tools are available today [2-7]. An effort is also in progress, through a Spanish initiative, to obtain the whole genome sequence of this crop [8]. These tools are generating a lot of information about genes involved in various biological processes, such as plant resistance and fruit quality [9,10]. However, the tools necessary for reverse genetic studies to conduct the functio
Mutation detection using ENDO1: Application to disease diagnostics in humans and TILLING and Eco-TILLING in plants
Karine Triques, Elodie Piednoir, Marion Dalmais, Julien Schmidt, Christine Le Signor, Mark Sharkey, Michel Caboche, Bénédicte Sturbois, Abdelhafid Bendahmane
BMC Molecular Biology , 2008, DOI: 10.1186/1471-2199-9-42
Abstract: The co-agroinfiltration of ENDO1 and p19 constructs into N. benthamiana leaves allowed high level of transient expression of a mismatch-specific and sensitive endonuclease, ENDO1 from Arabidopsis thaliana. We demonstrate the broad range of uses of the produced enzyme in detection of mutations. In human, we report the diagnosis of the G1691A mutation in Leiden factor-V gene associated with venous thrombosis and the fingerprinting of HIV-1 quasispecies in patients subjected to antiretroviral treatments. In plants, we report the use of ENDO1 system for detection of mutant alleles of Retinoblastoma-related gene by TILLING in Pisum sativum and discovery of natural sequence variations by Eco-TILLING in Arabidopsis thaliana.We introduce a cost-effective tool based on a simplified purification protocol of a mismatch-specific and sensitive endonuclease, ENDO1. Especially, we report the successful applications of ENDO1 in mutation diagnostics in humans, fingerprinting of complex population of viruses, and in TILLING and Eco-TILLING in plants.Scanning DNA sequences for mutations and polymorphisms is an analytic tool in a broad range of disciplines. However, identification of such mutations and polymorphisms in long stretches of DNA and in large numbers of samples by direct sequencing is not a trivial exercise. Several mutation detection techniques based on the physical properties of single stranded DNA or heteroduplex DNA [1-5] have been described. Among such methods are conformation sensitive gel electrophoresis (CSGE) [3], denaturing gradient gel electrophoresis (DGGE) [6], constant denaturing capillary electrophoresis, (CDCE) [7], Temperature Gradient Capillary Electrophoresis (TGCE) [8], single strand conformation polymorphism (SSCP) [2] and denaturing high-performance liquid chromatography (DHPLC) [1]. Other methods exploit chemicals like groove binders or chemicals that cleave single strand DNA at the mismatch site in heteroduplex DNA [9].Single strand specific nucleases
EcoTILLING for the identification of allelic variants of melon eIF4E, a factor that controls virus susceptibility
Cristina Nieto, Florence Piron, Marion Dalmais, Cristina F Marco, Enrique Moriones, Ma Luisa Gómez-Guillamón, Verónica Truniger, Pedro Gómez, Jordi Garcia-Mas, Miguel A Aranda, Abdelhafid Bendahmane
BMC Plant Biology , 2007, DOI: 10.1186/1471-2229-7-34
Abstract: A collection of Cucumis spp. was characterised for susceptibility to MNSV and Cucumber vein yellowing virus (CVYV) and used for the implementation of EcoTILLING to identify new allelic variants of eIF4E. A high conservation of eIF4E exonic regions was found, with six polymorphic sites identified out of EcoTILLING 113 accessions. Sequencing of regions surrounding polymorphisms revealed that all of them corresponded to silent nucleotide changes and just one to a non-silent change correlating with MNSV resistance. Except for the MNSV case, no correlation was found between variation of eIF4E and virus resistance, suggesting the implication of different and/or additional genes in previously identified resistance phenotypes. We have also characterized a new allele of eIF4E from Cucumis zeyheri, a wild relative of melon. Functional analyses suggested that this new eIF4E allele might be responsible for resistance to MNSV.This study shows the applicability of EcoTILLING in Cucumis spp., but given the conservation of eIF4E, new candidate genes should probably be considered to identify new sources of resistance to plant viruses. Part of the methodology described here could alternatively be used in TILLING experiments that serve to generate new eIF4E alleles.Plant viruses are obligate parasites that infect plants owing to specific interactions between virus and host factors that determine the plant susceptibility to viral infection [1,2]. Mutation or loss of one such susceptibility factor may result in virus resistance. Therefore, genes encoding susceptibility factors constitute potential targets for biotechnological and genomics-assisted breeding for improvement of crops resistance to viruses [3]. Throughout the last decade several susceptibility factors to plant viruses have been identified and characterized using model organisms as experimental systems [4-6]. However, among these factors, only translation initiation factors of the 4E family (eIF4E and eIF [iso]4E) and eIF4 [
Development of a Cucumis sativus TILLinG Platform for Forward and Reverse Genetics
Adnane Boualem, Sebastien Fleurier, Christelle Troadec, Pascal Audigier, Anish P. K. Kumar, Manash Chatterjee, Abdullah A. Alsadon, Monther T. Sadder, Mahmoud A. Wahb-Allah, Abdullah A. Al-Doss, Abdelhafid Bendahmane
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0097963
Abstract: Background Cucumber (Cucumis sativus) belongs to the Cucurbitaceae family that includes more than 800 species. The cucumber genome has been recently sequenced and annotated. Transcriptomics and genome sequencing of many plant genomes are providing information on candidate genes potentially related to agronomically important traits. To accelerate functional characterization of these genes in cucumber we have generated an EMS mutant population that can be used as a TILLinG platform for reverse genetics. Principal Findings A population of 3,331 M2 mutant seed families was generated using two EMS concentrations (0.5% and 0.75%). Genomic DNA was extracted from M2 families and eight-fold pooled for mutation detection by ENDO1 nuclease. To assess the quality of the mutant collection, we screened for induced mutations in five genes and identified 26 mutations. The average mutation rate was calculated as 1/1147?Kb giving rise to approximately 320 mutations per genome. We focused our characterization on three missense mutations, G33C, S238F and S249F identified in the CsACS2 sex determination gene. Protein modeling and crystallography studies predicted that mutation at G33 may affect the protein function, whereas mutations at S238 and S249 may not impair the protein function. As predicted, detailed phenotypic evaluation showed that the S238F and the S249F mutant lines had no sexual phenotype. In contrast, plants homozygous for the G33C mutation showed a complete sexual transition from monoecy to andromonoecy. This result demonstrates that TILLinG is a valuable tool for functional validation of gene function in crops recalcitrant to transgenic transformation. Conclusions We have developed a cucumber mutant population that can be used as an efficient reverse genetics tool. The cucumber TILLinG collection as well as the previously described melon TILLinG collection will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in cucurbits in general.
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