PhD thesis in progress
- PhD thesis staff: Philippe Lashermes
- Date of PhD thesis defense: 2015
"Early stages of the actinorhizal symbiosis : functional characterisation of the genes Cg12 and CgZF1"
- PhD thesis staff: Diaga Diouf and Antony Champion
- Date of PhD thesis defense: August 2014
Tropical trees from the Casuarinaceae family are able to colonise poor or degraded soils due to symbiotic interactions with soil microorganisms. In particular, they can enter nitrogen-fixing root nodule symbiosis with the soil bacterium Frankia. The general aim of this PhD project is to understand the molecular mechanisms involved in the symbiotic interaction between C. glauca and Frankia. The objective is to identify and characterize plant genes involved in the symbiotic infection process.
« Lipid metabolism in the developing argan seed »
- PhD thesis staff: Meriem Kaid-Harche (Université des Sciences et de la Technologie d’Oran) and Stéphane Dussert (UMR Diade)
- Date of PhD thesis defense: December 2014
The argan tree (Argania spinosa L.) is endemic to Morocco and to the Algerian region of Tindouf. The natural argan forest is exploited by local communities for many uses, including the production of argan oil. It is extracted from seeds (‘kernels’) and is used locally for food, cosmetic and medical purposes. Because it is exceptionally rich in vitamins E (tocols) and antioxidants, there is also a growing demand from the cosmetic industry for argan oil. Though the chemical composition of argan oil is now well characterized, lipid metabolism in the developing argan seed has been very poorly studied so far. It is an albuminous seed which constitutes an original model since the embryo and the endosperm are both lipid rich and of equal mass. The first objective of this PhD study will consist of characterizing the lipidome of both tissues using analytical chemistry techniques. The anatomy and the histology of the developing argan seed, embryo and endosperm will be then described, and the lipid classes that display significant differences between the endosperm and the embryo of the mature seed will be further characterized throughout seed development. The molecular determinants involved in tissue-specific biochemical features will be investigated through the comparative analysis of the transcriptome of the developing embryo and endosperm.
- PhD thesis staff:
- Date of PhD thesis defense : 30 septembre 2015
"Identification and analysis of the regulatory components underlying the fruit abscission processes of oil palm (Elaeis guineensis Jacq.)"
- PhD thesis staff: J.L. Verdeil and T.J. Tranbarger
- Date of PhD thesis defense: August 2015
Background and Context
The shedding of plant organs is a highly coordinated, developmentally regulated event that can occur in different contexts throughout the plant life cycle. Organ shedding is important for both plant vegetative and reproductive development, including abscission of leaves, branches, whole flowers, floral parts, seeds and immaturely aborted or ripe fruit. In particular, cell separation that occurs during fleshy fruit abscission and dry fruit dehiscence facilitates seed dispersal, the final stage of reproductive development, and therefore governs important characters in many crop species. For fruit to be shed, cell separation must occur in a precise location timed to optimize dispersal under the most favorable conditions. For crop species, if fruit are shed too early or late, economic consequences can be significant. Whereas our understanding of the evolutionary context for this phenomenon is mainly limited to model systems such as tomato and Arabidopsis, less is known about the mechanisms underlying fruit abscission in non-model crop species in general and, monocot species in particular.
Oil palm is a tropical perennial monocotyledonous species in the family Arecaceae with an extraordinarily oil rich fleshy mesocarp, which is the number one source of edible vegetable oil worldwide. One limitation is the difficulty to schedule regular harvests due to non-synchronized fruit shedding which results in a labor-intensive logistics that increases overall production costs. Furthermore, a two-stage process involving primary and adjacent abscission zones (AZs), plus the extraordinary low amount of methylated pectin and high levels of polygalacturonase (PG) activity, suggest divergent mechanisms may underlie the cell separation process that leads to fruit shedding in this monocotyledonous species. These and other recently identified original characters of oil palm fruit shedding warrant further detailed investigations.
Objectives and Approaches
The present study has the objective of determining the regulatory components underlying oil palm fruit shedding.
1. Mine available transcriptome and proteome data via bioinformatics to identify regulatory genes (e.g. transcriptional factors and signaling components) that are co-regulated during the abscission process based on approaches developed in the BDP group (Tranbarger et al., 2011).
2. Validate expression profiles of selected candidates via qPCR
3. Isolate and analyse promoter sequences of selected candidates to search for conserved and/or novel regulatory elements compared with model species. The oil palm genome sequence will be made public during the early to mid part of the thesis, the availability of which will facilitate the promoter analysis.
4. Functional validation studies (e.g. transient protoplast expression or stable expression in heterologous model systems such as tomato or Arabidopsis).
"Molecular determinants of the Arecaceae fruit and seed oil fatty acid composition"
- PhD thesis staff: S. Dussert and F. Morcillo
- Date of PhD thesis defense: décember 2015
Vegetable oils play a major economic and social role in both Western and developing countries. The usage of a given oil in food or in an industrial context (e.g. detergents, cosmetics, biofuels) is determined mainly by its fatty acid (FA) composition. The mechanisms that determine the considerable variability in lipid FA composition seen in the seeds and fruits of plants remain poorly understood. Oils extracted from the fruit mesocarp and seed of cultivated palms (oil palm, coconut ...) are the main world sources of palmitic acid and lauric acid respectively. The world's industrial demand for oils rich in these two FAs is undergoing spectacular growth. However, substantial differences exist among palms with regard to the FA composition of lipids accumulated in the mesocarp and the seed. The present work is aimed at identifying the molecular determinants that govern this interspecific variability. Two levels of diversity will be explored. Within the genus Elaeis, the present study will use a back-cross population between the African species E. quineensis and its American relative E. oleifera. Then, in the second step, about one hundred species of the family Arecaceae will be investigated. In both cases, a recently developed systems biology approach, involving the analysis of gene co-expression networks, will be used after obtaining transcriptomic and lipidomic datasets of the studied tissues throughout their development.
"Search for early molecular markers of the mantled floral variation of oil palm"
- PhD thesis staff : Estelle Jaligot and Alain Rival
- Date of PhD thesis defense: Decembre 2015
- identifying expression markers of the mantled somaclonal variation through the comparison between the true-to-type and the variant transcriptome.
- assessing the discriminating power of the selected markers at early stages of the in vitro process.
Strategy and Methods :
- Transcriptomic analysis of the normal oil palm inflorescence, construction of a reference transcriptome. Technique : RNAseq, Illumina sequencing
- Identification of sequences and pathways of interest. Technique : bioinformatic analysis of sequencing data.
- Comparison between the normal and the mantled inflorescence transcriptome through the re-sequencing of libraries generated from several different clonal lines. Technique : Illumina.
- Identification of sequences displaying consistently a phenotype-dependent differential expression pattern. Technique : bioinformatic analysis of sequencing data, statistical analysis of expression patterns.
- Validation of candidate markers on normal/mantled regenerant palm pairs from different clonal lines and on normal-/mantled-derived in vitro cultures at various stages of the industrial regeneration process. Technique : quantitative PCR (q-PCR).
ISSAKA SALIA Ousseini
- PhD thesis staff : Dr Yves Vigouroux, Institut de Recherche pour le Développement, UMR DIADE, Montpellier, France., Dr Kane Ndjido, Institut Sénégalais de Recherches Agricoles, ISRA, LNRPV, Dakar, Sénégal. et Dr Yakoubou Bakassou, Faculté des sciences, Université Abdou Moumouni, Niamey, Niger.
- Date of PhD thesis defense : November 2015
"Adaptation de l’architecture racinaire du mil :Phénotypage et analyse spatio-temporelle de systèmes racinaires en croissance"
- PhD thesis staff: Laurent Laplaze
- Date of PhD thesis defense : August 2016
"Salt tolerance in the tropical trees of the Casuarinaceae family"
- PhD thesis staff: Laurent Laplaze, Ibrahima Ndoye (UCAD) and Valérie Hocher
- Date of PhD thesis defense: 2015
Soil salinisation is a major problem worlwide that could lead to the loss of 50% of arable lands by 2050. In Senegal, 1,2 of the 3,8 millions acres of cultivated lands are affected by salinisation. Tropical trees from the Casuarinaceae family are largely used in tropical and subtropical areas to rehabilitate degraded soils. They are pioneer species able to colonise poor soils due to symbiotic interactions with soil microorganisms. Some species are also tolerant to salt. The objective of this PhD project is to characterize the mechanisms responsible for salt tolerance in Casuarina glauca . In particular, changes in transcriptome induced by salt treatments will be analysed using next generation sequencing (RNA-seq) in the framework of a project funded by the Joint Genome Institute (JGI, USA). This work is conducted in collaboration with the University of New Hampshire (USA), the Institute of Forest Genetics and Tree Breeding (India) and the Laboratoire Commun de Microbiologie IRD/ISRA/UCAD (Senegal).
"Identification of resistance genes to Rice yellow mottle virus (RYMV) in the African cultivated rice species, O. glaberrima"
- PhD thesis staff: Alain Ghesquière et Laurence Albar
- Date of PhD thesis defense : 2016
In Africa, rice is a major staple food in many countries and Rice yellow mottle virus (RYMV) is one of its main biotic constraints. The development of sustainable resistance against RYMV is therefore an important challenge for rice breeding and people in Africa. A first resistance gene, RYMV1, encoding a translation initiation factor, has been previously identified and well characterized in the lab but the emergence of RYMV genotypes able to overcome this resistance gene has been demonstrated in laboratory conditions. Expanding the number of resistance genes is a prerequisite to increase the durability of resistance of rice at field level. Fortunately, additional resistance sources are known in the cultivated African rice species, O. glaberrima, but little information is still available on the genes underlying resistance. The aim of the project is to identify genetic factors involved in RYMV resistance using two complementary approaches, and to characterize the most important ones.
A first approach will focus on two highly resistant O. glaberrima accessions, Tog7291 and Tog5307, whose resistance is under distinct and original genetic determinisms compared to RYMV1 one. This approach will involve family-based linkage analysis and NGS sequences as a source of mapping resistance genes and determination of molecular markers. The main output will be the identification of the genetic basis of resistance (number of genetic factors, mode of inheritance) and mapping of resistance genes. Depending of the results, fine mapping and candidate gene characterization will be undertaken on the most important loci. Functional characterization of the gene RYMV2 in Tog7291 accession and recently identified in the lab resistance will be completed.
A second approach will use deep sequencing data to explore the diversity in an available African rice collection (including wild and cultivated species) to identify new resistance genes or QTLs based on an association mapping analysis, and to identify original alleles of RYMV1 and RYMV2 resistance genes. This approach will consider both high and partial resistance
"Use of cryopreservation for the conservation and production of date palm (Phoenix dactylifera) in vitro cultures
Impact of cryopreservation on several biological parameters of cultures"
- PhD thesis staff: Florent Engelmann
- Date of PhD thesis defense: 2015
The date palm plays a key role in the ecology of arid and desert zones of the Sahara, the Middle East, the Arabian Peninsula, as well as Iran and the Indus Valley in Pakistan. It has a very high economic and sociological value for the populations of these regions, both through its own products (dates and other products) and because it allows the development of oasis agriculture.
The conservation and production of this species faces problems related to its biology. For conservation, date palm seeds are classified as orthodox and their viability is very long. However, these seeds are heterozygous and are therefore only of limited interest because they allow the conservation of genes but not of specific genotypes. Another problem is the multiplication of selected individuals, which is limited by the low number of suckers produced during the life of a date palm.
The use of biotechnology allows overcoming these difficulties. In vitro culture (somatic embryogenesis) allows the mass production of clones from selected individuals. This technique is now widely applied in many industrial laboratories. However, it is necessary to conserve the clones established because of the risk of somaclonal variation, which increases with the number of culture cycles, and to optimize the management of crop production. Cryopreservation (liquid nitrogen, -196 ° C) is the only technique currently available for long-term, safe and cost-effective conservation of in vitro grown plant material. Cryopreservation protocols have now been developed for several hundred plant species. However, studies aimed at understanding the biological and biophysical mechanisms involved during a cryopreservation protocol are still limited.
A highly efficient cryopreservation protocol based on the droplet-vitrification method was recently developed for proembryogenic masses (Fki et al. 2011). Its application has enabled long-term preservation of both commercial and local varieties, often threatened by the widespread commercial varieties.
Proposed research topics
The research project aims at improving our understanding of the impact of successive steps of the cryopreservation protocol used on the physiology of date palm in vitro cultures, using different approaches.
The proembryogenic mass cryopreservation protocol developed by our collaborators in Tunisia is used as experimental model for this study. We will select treatments leading to high, intermediate or low / zero survival, i.e. weel controlled and contrasted conditions. Using this experimental tool, we will study the impact of the successive phases of the protocol using different approaches / tools:
- histo-cytological approach: We will study the effect on the structural integrity of tissues of dehydration with cryoprotective solutions (loading solution, vitrification solution, unloading solution) and liquid nitrogen exposure by performing a qualitative and quantitative histological study. We will also observe the effect of these treatments on the rate of recovery. We will use classical histology techniques, allowing a qualitative assessment, but also image analysis techniques, which allow a quantitative assessment of the effect of treatments. We will also study the dynamics of action of treatments with cryoprotective solutions at the level of the whole explant and at the cellular level, using the real time microscopy (RTM), which has so far been used only on a very small number of plant models.
- molecular approach : two main aspects will be addressed. We will study gene expression during different stages of the cryopreservation protocol and during regeneration of plantlets using transcriptomics techniques. We will also measure their effect on the intensity of DNA methylation.
The research will be performed in IRD Montpellier in the DESSITROP team (Tolerance to dessiccation and conservation of biodiversity of tropical and mediterranean plant species), which bélongs to the UMR DIADE, Under the responsibility of Florent Engelmann, Director of research in IRD, Head of the DESSITROP team. It will include strong collaborations with various partners in Montpellier (cellular imagery platform in CIRAD, palm development team in the UMR DIADE) and in Tunisia. A training period in in vitro culture and cryopreservation techniques will be organized for Mohammad Salma at the beginning of the thesis. Regular visits of Tunisian colleagues in Montpellier are also planned.
" Biosystemactic study of the genus Craterispermum (Rubiaceae) in continental Africa"
- PhD thesis director: Bonaventure Sonke (Université de Yaoundé I, Cameroun), Perla Hamon and Petra de Block (Jardin Botanique National de Belgique)
- Date of PhD thesis defense: December 2013
A taxonomic revision of the genus Craterispermum in continental Africa was carried out after which 19 taxa (including 16 species and three varieties) were recognized. Eight new species and three varieties were described. The most important distinctive characters are vegetative (stipules, young branches, leaves) and inflorescence characters. According to IUCN criteria, 38% of the taxa are threatened by extinction in the wild. Morphometric analysis of the complex C. cerinanthum, C. laurinum and C. schweinfurthii shows that C. laurinum is rather well delimited, as well by its characters as by its distribution strictly confined in the western part of the Upper Guinea Domain. Taxonomic overlap between C. cerinanthum and C. schweinfurthii remains high in central Africa, where “typical” spécimens mix with atypical ones. Pollen of Craterispermum are (2-)3(-4)-zono-colporate or -porate. Pollen of the brevistylous morph is characterized by the presence of supratectal elements, whereas the nexine of pollen of the longistylous morph is smooth. The systematic value of pollen characters is low at species level. The compound apertures seem to consist of only two strata. Craterispermum is distributed in continental Africa from Senegal to Mozambique. The Lower Guinea Domain is recognized as the principal center of diversity and endemism of Craterispermum in continental Africa. Despite the fact that the genera Coffea, Craterispermum and Hedyotis are not closely related, their respective genomes show similar conserved regions. Methods, approaches and tools developed for Hedyotis and Coffea can be used to study the genome of Craterispermum. Amplification tests show that 14 microsatellite markers derived from coding sequence libraries (EST) of Coffea and Hedyotis are useful in genetic diversity analyses in Craterispermum. The estimated values of the genome sizes in this genus vary between 1.03 pg and 1.64 pg. Dry material (silica gel and herbarium) can be used in flow cytometry if a number of conditions is respected. Genome size can be used to test systematic hypotheses in Craterispermum.
Keywords: Revision, taxonomy, palynology, microsatellite markers, Craterispermum, Rubiaceae.
« Etude des variations des rythmes de croissance et élaboration d’un modèle ontogénique et architectural du palmier-dattier (Phoenix dactylifera L.) : cas du Niger au Sahel.»
- PhD thesis staff : de Reffye Philippe (AMAP-CIRAD) et Bakasso Yacoubou (FST-UAM), Hervé Rey (CIRAD-AMAP) and Jean-Christophe PINTAUD (DIADE-IRD)
- Date of PhD thesis defense : 25 septembre 2016
The current climate change imposes strong constraints substantially alters the phenology of cultivated varieties and compromises the performance of some elite varieties. This change takes place simultaneously with the rapid population growth in the Sahel countries. Consequently, these countries face to chronic food insecurity, hence the interest to focus on the date palm (Phoenix dactylifera L.), tolerant species of harsh conditions in arid areas, which can contribute to food security. This PhD project aims to study the growth rates and architectural different parameters of the reproductive organs of the date palm in order to develop an ontogenetic and architectural model. The purpose of this model is to move towards a functional model that could contribute to the selection of cultivars to improve the resistance of the species and especially the production of dates.
Keywords:Phoenix dactylifera, growth, modeling, architecture, production, dates
PhD thesis defended
"Date palm diversity in Pakistan and its relationship with world dates germplsm - exploring the centre of origin of Phoenix dactylifera L."
- PhD thesis staff: Iqrar A. Khan (Vice Chancellor, University of Agriculture Faisalabad-Pakistan ), Jean Christophe Pintaud
- Date of PhD thesis defense: December 2013
Date palm is third major fruit crop after citrus and mango in Pakistan. The origin and domestication history of the date palm (Phoenix dactylifera L.) remains obscure. The present study will be conducted to identify variations among available date palm genepool in Pakista through seed morphometrics and molecular tools. It will be compared with regional date palm germplasm. To reveal the variations among available date palm gene pool, molecular analyses will be carried out using SSR markers. To explore the history and evolution of date palm, seed morphometric technique will be used.
" Flavonoids and actinorhizal symbiosis : RNA interference silencing of chalcone synthase gene in Casuarina glauca plants"
- PhD thesis staff: Valérie Hocher and Didier Bogusz
- Date of PhD thesis : September 2012
Actinorhizal root nodules result from the interaction between a nitrogen-fixing actinomycete called Frankia and roots of dicotyledonous plants belonging to 8 plant families and 25 genera. Most actinorhizal plants are capable of high rates of nitrogen fixation comparable to those found in legumes. As a consequence, these plants are able to grow in poor and disturbed soils and are important elements in plant communities worldwide. In addition, some actinorhizal species can grow well under a range of environmental stresses such as high salinity, heavy metal and extreme pH. This facility for adaptation has drawn great interest to actinorhizal plants, particularly to several species of Casuarinaceae such as Casuarina glauca , which can be used for fuelwood production, agroforestry, and land reclamation in the tropics and subtropics. The basic knowledge of the symbiotic association between Frankia and actinorhizal plants is still poorly understood, although it offers striking differences with the Rhizobium -legume symbiosis. The rhizogenesis group (IRD, Montpellier, UMR 188) has concentrated on the molecular study of the plant genes involved in the interaction between Frankia and the tropical tree Casuarina glauca .
Flavonoids are secondary metabolites derived from the phenylpropanoid pathway. They play key roles at different levels of plant microbe interactions especially in the symbiosis interactions. In legumes specific flavonoids released from the roots interact with the nodD protein of Rhizobium to activate transcription of other nod genes responsible for the synthesis of nod factors Recent studies using RNAi-mediated inactivation of key flavonoid genes confirmed the essential role of flavonoid for legumes nodulation (Wasson et al ., 2006). In actinorhizal plants, the involvement of flavonoids in symbiosis is poorly understood, but because of the similarities of the infection process between some actinorhizal plants and legumes, flavonoids were proposed to act as plant signals activating the production of Frankia root hair deforming factor.
The objectives of this thesis are to understand the role of flavonoids during actinorhizal symbiosis. We propose to use the actinorhizal model system C. glauca and its symbiotic partner Frankia . Our experimental approach is based on RNAi technology and is similar to that developed on M. truncatula by Wasson et al . (2006). Chalcone synthase (CHS ) is the enzyme that catalyzes the first committed step of the flavonoid pathway. We propose to silence the chalcone synthase gene in C. glauca using the recent RNAi methodology developed in our team (Gherbi et al ., 2008). Effect of chalcone synthase silencing will be observed on plant during nodulation. QPCR control will be used to control gene expression knock down and biochemical assays will be done to analyse and measure flavonoid content of silenced plants.
"Genetic mapping and study of the interspecific cross between Coffea pseudozanguebariae Bridson and Coffea canephora Pierre."
- PhD thesis staff: Perla Hamon et Arsène Zoro Bi (Université Nangui Abrogoua)
- Date of PhD thesis : December 2012
Among the Coffea genus, only two species Coffea arabica (ARA) and Coffea canephora (CAN well known as Robusta) are cultivated over the world. This latter is the only Coffea species cultivated in Ivory Coast. Unfortunately, the high caffeine content (2.5% dmb) and the bitterness of the coffee beverage for CAN compared to ARA depreciate its commercial value. The wild species C. pseudozanguebariae (PSE), naturally caffeine-free, gives the possibility for the selection of new introgressed varieties of C. canephora with no or low caffeine content. F1 hybrids and BC1 progeny were produced from PSE X CAN and (PSE X CAN) X CAN crosses respectively at the CNRA research station in Man, Ivory Coast. These plants can now flower and produce seeds. In this study we analyzed a set of traits important for cultivated varieties such as the phenology of flowering, the number of flowers per node (NFN), the fructification duration (FD), the pollen viability (PV), the rate of fructification (RF), the seed-set (SSET), the weight of 100-seed (W100) and we estimated the caffeine (CAF) and heteroside contents (RHET). Moreover, the BC1 progeny was genotyped using molecular markers (AFLPs and SSRs). The genetic map produced was used to identify and to map several QTLs for some traits of interest (PV, NFN and FD).
The results showed that all the F1 individuals as well as 80% of the BC1 flower lately (in February for Ivory Coast conditions) as the wild parent (PSE). While the remaining 20% of the BC1 flower early (in January) like as the cultivated parent (CAN). The fertility and the productivity of the F1 hybrids were both very weak: on average, depending on the year, 5 to 13% of pollen were viable; the average fructification rate was 9.6% with a mean of seven flowers per node. An average of one seed per fruit and a 100-seed weight of 8 grams were obtained. Unfortunately, the fertility was not restored in BC1 as well as expected. However, an important variability was observed between individuals.
Compared to the parents, the F1 genotypes had intermediate foliar values (0.9 cm for petiole length, 13.4 cm for leaf length and 5.9 cm for leaf width). Their fructification duration (FD) was on average 175.5 days and all the fruits were purple-dark similarly to the wild parent (PSE). Seed caffeine content was on average 0.36% dmb and the heteroside relative value was of 0.44. Regarding the BC1 progeny, the variability was very important for all the traits, with generally average values comprised between F1 and CAN estimations.
Genetic studies revealed that the mature fruit color (MFRCOL) is controlled by a single gene with two alleles (R/r), R (purple-dark) being dominant allele over r (red). Short vs. long FD is controlled by a major gene (fd) with two co-dominant alleles (fd1 and fd2). The presence vs absence of caffeine, as well as the presence vs absence of heteroside, are controlled by a major gene with two co-dominant alleles (het1 and het2) for the heteroside whereas for caffeine, the presence (caf2) was dominant over the absence (caf1). The variations, between genotypes characterized with short or long FD and between genotypes containing caffeine or heteroside, are determined by genes with quantitative effects. Foliar dimensions (FOL), PV, NFN and W100 were all under a polygenic control. Genes effects were additive for FOL, FD, RHET and W100. Nevertheless, there was a deviation for CAF, NFN and PV. The major genes fd and CAF are genetically linked while they were both independent to het1/het2 and R/r couples.
The relationships between these characters highlighted two main factors. The first one, which could be considered as the production, was defined by the following variables: FOL, FD, W100, CAF, NFN and MFRCOL. They were all significantly and positively correlated and opposed to RHET. The second factor, defined by variables RF, SSET and PV, corresponded to the fertility.
The inter-specific genetic map, covering a genetic distance of 1955.4 centimorgans, remained unsaturated. Indeed, 19 linkage groups were found instead of the expected 11. Nevertheless, a total of 11 QTLs were identified. Among them, five (four QTLs for VP and one for NFN) were mapped while one for VP and one for FD co-segregated with unlinked markers.
"Identification and Validation of molecular markers associated with resistance to Striga hermonthica (Del.) Benth. and Initial characterization of resistance mechanisms in pearl millet [Pennisetum glaucum (L.) R. Br.]"
- PhD thesis staff: Dominique This, Pr. Toudou ADAM (University of Niamey, Niger), Pr. Ibrahim A. AMOUKOU (University of Niamey), Dr. C.T. Hash, Dr. Bettina I.G. Haussmann (ICRISAT-Niamey) and Yves Vigouroux (IRD)
- Date of PhD thesis defense: November 2013
Striga hermonthica (Del.) Benth. is a major biotic constraint to grain production of pearl millet in Niger, a West African country characterized by high population growth and where grain yields are very low (about 400-600 kg ha-1). In tropical regions prone to parasitism, yield losses due to Striga can reach 100% because of the lack of resistance in cultivars and particularly under drought conditions. Breeding for Striga resistance in cultivated pearl millet is still challenging. However, it is now necessary to identify genomic regions conferring resistance to Striga in pearl millet. Our objective is therefore the identification and validation of QTLs and the characterization of mechanisms of resistance to Striga. The identification of the genetic basis of this resistance will then strengthen the resistance of varieties of millet. Molecular tools are now available from millet and have proven effective in other species including sorghum, rice, maize and cowpea. This progress should accelerate the development of resistant cultivars through the adoption of an appropriate strategy for marker-assisted selection (MAS). The genetic control through use of resistant cultivars offers a practical and economically feasible, either independently or as a central component of a comprehensive integrated Striga control approach.
"Population genetics and architecture and production plant models. Application to the Mascarocoffea genetic resources preservation."
- PhD thesis staff: Philippe de Reffye, Sylvie Sabatier (AMAP-Montpellier), Perla Hamon (DIADE-Montpellier), Jean-Jacques Rakotomala (FOFIFA-Madagascar)
- Date of PhD thesis : December 2012
Mascarocoffea species (61 / 124 described, endemics to Madagascar) are highly endangered because of the considerable Madagascar area forest reduction. For their preservation, assessment of the Mascarocoffea diversity level in the FOFIFA Kianjavato Collection Research Station compared to four in situ populations was made. The collection population’s diversity level is larger than that in situ, observed heterozygosity is similar with a significant allelic richness. Modeling the young individuals in these populations’ growth and development by the GreenLab model helped to understand the development of the plant structure in accordance with the interspecific architectural variability in studied populations at different development stages. A good fitting on growth was obtained on the populations studied in the experimental plot. The evolution of the GreenLab model key variable, the source-sink ratio (Q / D), showed that the production of many more organs influences the ratio Q / D evolution in time. This variable affects organ size and Mascarocoffea architecture. An essay on adult individuals growing in different environments (in and ex situ), whose morphological, genetic and architectural parameters were determined in this study, would allow the plants response in terms of architecture depending on the environmentin which they grow and to optimize the model. The detection of hybrid individuals in both in situ and at the collection would detect hybrids characters on the plant structure and consider integrating genetic parameters in the model....
Keywords : genetic diversity ; growth ; Madagascar ; architecture variability ; Greenlab ; model ; Mascarocoffea
“Identification of genes involved in lead tolerance in the brassicaceae Hirschfeldia incana"
- PhD thesis staff:
- Date of PhD thesis : December 2012
Exploited, and then abandoned, mining sites, in the oriental Morocco, have polluted the neighboring regions, by dispersion of mining wastes containing Pb. Facing this problem, it is necessary to develop phytomanagement programs. Nevertheless, these project realisations encountered two major constraints: weak metallophyte plant diversity and misunderstanding of Pb tolerance mechanisms.
In this context, the objectif of my work was to select plants compatible with a phytoremediation program and to identify genes implicated in lead tolerance.
Firstly, soil and mining waste analysis has revealed an important polymetallic pollution of the studied region. Then, floristic explorations had permitted to identify a plant of the brassicaceae family, Hirschfeldia incana, which is predominant on the polluted mining regions and accumulated Pb. Pb accumulation in H. incana was confirmed in controlled conditions and a candidate gene approach has shown the lead tolerance implication of two genes, one coding for a metallothionein (MT2A) and the other a membrane transporter (HMA4). Finally, the transcriptome comparison from H. incana and A. thaliana has generated a list of candidate genes putatively implicated in Pb tolerance. Functional analysis of four genes, coding a ferretin, a metallothionein, a copper binding protein and a defensin, has permitted to clarify their role in Pb tolerance.
Key words: Hirschfeldia incana, lead (Pb), tolerance, mining wastes, pollution, phytoremediation, Arabidopsis thaliana, transcriptome, Morocco
"Molecular study of the early stages of actinorhizal symbiosis Casuarina-Frankia: functional analysis of genes controlling the host plant infection"
- PhD thesis staff: Claudine Franche, Nadia Ykhlef and Hassen Gherbi
- Date of PhD thesis : June 2012
The goal of the thesis project is the functional characterization of the CgCCaMK gene (Calcium & Calmodulin-dependant kinase) recently isolated from Casuarina. CgCCaMK is the homologue of the MedicagoMtDMI3 (CCaMK). CCaMK is supposed to decode calcium oscillations that occur during early stages of the symbiotic interaction. The deduced protein contains a calmodulin-binding domain that is also an autoinhibitory domain. The putative role of CgCCaMK during Frankia infection processus and the importance of the autoinhibitory domain in the nodule ontogenesis will be studied.
"Sexual differentiation in palms: a comparative study of dioecious and monoecious taxa in the tribe Chamaedoreeae"
- PhD thesis staff: James Tregear (in collaboration with G. Galeano/X. Marquinez, UN Bogotá and F. Stauffer, CJB Geneva)
- Date of PhD thesis defense: November 2014
Approximately 10% of flowering plant species are sexually dimorphic, ie. producing at least some unisexual flowers. Male and female flowers may be produced either on the same plant (monoecious species) or on separate plants (dioecious species). Plant breeding systems are of considerable importance in determining the yield of crop plants since fruits or seeds are often the product harvested. Moreover, they are of key importance in population genetics and evolution. The palm family (Arecaceae) is an interesting group of plants on which to focus studies of breeding systems and sexuality, since monoecy and doecy appear to have evolved independently on numerous occasions within this group, which includes many cultivated and wild species of importance to mankind. About 85% of palms produce single sex male or female flowers, either on dioecious or monoecious plants, yet very little is known of the mechanisms by which palms and their flowers acquire their male or female identity.
The general aim of this project is to contribute to a better knowledge of sex determination processes in palms by a comparative study between the monoecious and the dioecious genera of the tribe Chamaedoreeae. This group is of special interest because dioecy appears to have evolved independently on two separate occasions from a monoecious ancestor. Several major scientific questions are to be addressed in this project:
- What is the exact sequence of developmental events which results in the production of unisexual flowers in the dioecious palm genus Chamaedorea?
- How does this developmental sequence compare to that of a closely related monoecious species (in the genera Gaussia, Synechanthus or Hyophorbe) or to an independently evolved dioecious lineage (in the genus Wendlandiella)?
- What genes are likely to play a role in determining maleness or femaleness and is their role conserved throughout the tribe?
These questions are being addressed by a combined approach involving morpho-anatomical, transcriptomic and molecular histological techniques.
"Study of differential expression of the genome in relation to sex determination in the date palm (Phoenix dactylifera L.)
- PhD thesis staff: Frédérique Aberlenc, Alejandro Maass (Centre du Modélisation Mathématique, Université du Chili)
- Date of PhD thesis defense: November 2014
The date palm (Phoenix dactylifera L.) is a dioecious plant; the male and female flowers are borne on different individuals. Its sexual reproduction produces progeny of which only half of the individuals are females and produce fruits. However, given the length of its vegetative phase, it is necessary to wait six to eight years of first flowering to detect the sex of plants.
Knowing the sex of the date palm at a young age, would allow farmers to be able to select female plant reducing planting costs associated with the cultivation of unproductive male plants. Equally, early identification of sex would open new perspectives for the implementation of breeding programs by controlled crosses.
For this reason, understanding the cellular and molecular processes that control sex determination in the date palm is the main goal of the present study. To achieve this we will do a transcriptome analysis to identify genes expressed during flower development and build the signaling pathways involved in the process of sexual differentiation in this species.
- PhD thesis staff: Frédérique Aberlenc and Amel SALHI-HANNACHI, Salwa ZEHDI, LGMIB, Faculté des Sciences de Tunis
- Date of PhD thesis : April 2013
The rigorous separation of the sexes is widespread in animals in contrast, in plants: dioecy is very rare. Therefore differentiation and evolution of sex chromosomes is an important aspect of our knowledge of dioecious plants, notably for date palm. In this crop plant, only females are valuable since they produce dates. However, there is no way to identify the sex of date palm plants before reproductive age, and the sex-determining mechanism is still unclear. We thus conducted a large-scale population genetics survey, using simple sequence repeat (SSR) markers and identified three reliable sex-linked loci producing male specific alleles allowing us to identify the gender in 100% individuals. We confirmed thus the existence of an XY chromosomal system with a non-recombining XY-like region in the date palm genome. Furthermore a very low diversity associated with Y haplotypes is generated by these loci, which is consistent with clonal paternal transmission of a non-recombining male-determining region. The obtained results have established the date palm as a biological model with one of the most ancient sex chromosomes in flowering plants.
"Analysis of diversity and expression of active retrotransposons in Coffea species and cultivars"
- PhD thesis staff: Alexandre de Kochko, Claudia Carareto and Romain Guyot
- Date of PhD thesis defense: December 2014
The utilization of retrotransposons (RTs) as molecular marker contributes in the understanding of the organization and dynamic in large scale of the genome. Several characteristics make the RTs good molecular makers, as integration activity, persistence and dispersion in the genome, as well as, the presence of conserved motifs. The use these repeats as molecular marker has contributed for a better understanding of the evolutionary dynamics of genomes, especially the polyploidy genomes, and evolution of these transposable elements. The species of the genus Coffea stand out by economic importance and by wide variety of species and cultivars. This project aim at evaluating the use of active RTs, with different activity rates, as molecular marker in six Coffea species: the allotetraploid hybrid C. arabica and in its potential parental species, C. canephora and C. eugenioides; and three closer related species, C. heterocalyx, C. pseudozanguebariae and C. congensis. Additionally, it will be investigated C. arabica cultivars exposed to different stress conditions. For this, the selected RTs will have characterized both their structure and expression, and their availability molecular markers will be investigated by the molecular techniques of RBIP, SSAP, REMAP and IRAP. Then, the current project will contribute for characterizing active RTs in Coffea species, evaluate their genetic diversity, and understanding of probable effects of hydric stress on the RTs activity and, their functional and evolutionary dynamics in polyploid genomes.
Keywords: Retrotransposons; Coffea; molecular maker.
"Diversity of lead hyperaccumulation in Hirschfeldia incana: Prospects for phytoremediation."
- PhD thesis staff: Didier Bogusz and Yves Vigouroux
- Date of PhD thesis defense: December 2013
Lead (Pb) is a metal widely used in industry. However, it has a high toxicity to organisms. The extraction and lead processinggenerate large amounts of residues rich in heavy metals that cause problems for public health and the environment.
In order to identify resistant and/or accumulator plants of lead, which could be used for soil remediation, we conducted a survey in the mining sites of oued El Heimer, Touissit and Boubker (Region Oujda in eastern Morocco). Several samples of soil and plants were collected from 15 different stations and the determination of 7 heavy metals (Pb, Al, As, Cd, Cu, Ni and Zn) was performed by ICP.
The results showed that the soils contain high concentrations of heavy metals, mainly Pb and Zn. This indicates strong polymetallic pollution. An exploration of plant species growing on the contaminated areas was considered in the context of phytoremediation program. Among the various identified plants, Hirshfeldia incana, member of the Brassicaceae family, has been selected to study mechanisms of Pb tolerance.
For this species, a metallicolous population (M) from polluted sites (Oued el Heimer) and non metallicolous (NM) growing in unpolluted sites (Tetouan) were identified. The study of these different populations can provide some fundamental understanding of tolerance phenomena and hyperaccumulation of metals in plants.
The differences between M and NM populations will allow on one thehand to assess the degree of speciation in response to metal concentrations and to identify loci involved in tolerance and adaptation to heavy metal. On the other hand to choose the most appropriate for phytoremédiation strategies.
The thesis project has three complementary parts:
- Characterization of physiological tolerance and accumulation of lead in M and NM populations of H. incana.
- Intra- and inter-population analysis of sequences for candidate genes involved in tolerance and metal accumulation in plants.
- Validation of candidate genes by expression analysis and reverse genetics.
“Origins, evolutionary and biogeographical history of the date palm (Phoenix dactylifera L.) : the contribution of genetics and morphometrics”
- PhD thesis staff: JF Terral (Centre de Bio-Archéologie et d'écologie/UM2) and Jean-Christophe Pintaud
- Date of PhD thesis : December 2012
The objectives of this work are to understand the evolutionary history and biogeography of the date palm (Phoenix dactylifera L.), a species of importance to human populations in hot, arid regions of North Africa and the Middle East using genetic and morphometric analyzes. Indeed, the origins of the domestication of the date palm remain poorly understood despite the archaeological data that seem to attest its culture from the late 4th millennium BCE.
Phylogenetic analysis of the genus Phoenix made from chloroplast sequences identified close relatives of the date palm. Populations of wild date palms are recognized for the first time through analyses of genetic diversity and structure. Congruently, morphometric analysis of the seed outline based on the method of elliptic Fourier transform highlights a clear differentiation between wild and cultivated individuals. Characterization of changes in seed morphological traits related to domestication is used to define the status of wild or domesticated material excavated from various archaeological sites in Pakistan and Egypt. In addition, the date palm genetic study of various origins seems to indicate that at least two domestication events took place: one in Africa and one in the Middle East. Finally, genetic and morphometric studies, conducted separately or together depending on the type and age of the material analyzed, allow for the first time to discuss the origins, history and dynamics of date palm agrobiodiversity, in time and space.
"Functional analysis of S1 locus, the major factor of the reproductive barrier that separates the Asian and African cultivated species of rice."
- PhD thesis staff: Mathias Lorieux (IRD, Montpellier – CIAT, Colombie), Hélène Adam, Stéphane Jouannic (IRD, Montpellier)
- Date of PhD thesis defense: Décembre 2014
African rice species (O. glaberrima) is a valuable source of useful genes for rice improvement. However, hybrid sterility barrier between O. sativa and O. glaberrima hampers their whole exploitation. Candidate genes (F-box genes) at the S1 locus have already been identified as the main factors of this reproductive barrier, although validation of their function has not yet been confirmed. The principal aim of this thesis proposal is to determine the function of these recently identified factors in the S1 locus causing fTRD and female sterility in the F1 hybrids between Asian and African rice species. To achieve this goal, Knockout or down-regulation of F-box genes, detailedcytological examination of gametophytes, as well as phenotypic evaluation for sterility will be carried out. It is expected that identification, function and confirmation of S1 gene as responsible of female sterility in the F1 hybrids can be determined. The obtained results in the present project would be very useful to generate a better understanding of interspecific sterility barriers and also to derive strategies to better exploit African rice species in breeding programs.
"Common bases of the actinorhizal symbiosis: comparative analysis among Discaria trinervis and Casuarina glauca"
- PhD thesis staff: L. Wall and Sergio Svistoonoff
- Date of PhD thesis defense: September 2013
Nitrogen availability is commonly the major factor limiting the productivity of ecosystems around the world. In this context, biological nitrogen fixation mediated by actinorhizal symbiosis and legume-rhizobium symbiosis, plays a key role in the maintaining of ecosystems. Evolutionary analysis indicates that nitrogen fixing symbiosis evolved 50-100 million years ago (Kistner 2002). Phylogenetic analysis has shown that all plants able to developed nitrogen fixing nodules belong to a single clade: Rosid
Actinorhizal plants belong to 8 angiosperm families and 25 genera of angiosperms. All the host species are perennial woody shrubs or trees, with the exception of Datisca glomerata, which has herbaceous shoots. Actinorhizal plants are able to develop symbiotic root nodules after infection of their roots by filamentous gram-positive bacteria of the actinomycete genus Frankia (Wall 2000). The infection process in the roots of actinorhizal plant can occur in two different pathways: intracellular or intercellular (Wall and Berry 2008). In the first Frankia penetrates inside the root cell through a curled root hair, whereas the second mechanism involves an initial phase where the microsymbiont enter the root tissue through the intercellular spaces of the root tissue without penetrate inside the cells. Recently a model for the molecular evolution of infection pathways was proposed in legume, and could be extensive also to the actinorhizal symbiosis (Madsen 2010). In this model the intercellular infection pathway is suggested to be the ancient mechanisms of infection from which subsequent evolution process gave rise the crack entry and the more recent intracellular hair root infection pathways. In all cases the result is the formation of a novel host organ, the nodule, where Frankia differentiate in vesicles and the nitrogen fixation taking place.
Discaria trinervis is a South American actinorhizal shrub, that grows in western Argentina and eastern Chile, between 31° and 48° S. D. trinervis belongs to the Rhamnaceae family, which also includes Ceanothus spp. and other South American genera. In the field, the root system of D. trinervis is naturally nodulated by Frankia (Chaia 1997). A Frankia strain BCU110501 has been isolated from Discaria trinervis nodules (Chaia 1998) and has been well characterized, its genome is in progress, and it has been extensively used in infection and nodulation studies in actinorhizal plants (Wall 2000). The study of nodule development in D. trinervis reveals that the infection process in this actinorhizal plant follows the intercellular infection pathway (Valverde 1999). While many studies have been done in the intracellular infection pathway, little is known about the mechanisms involved in the intercellular pathway. Casuarina glauca is an actinorhizal tropical tree that develops nodules through the intracellular infection mechanism. C. glauca has been extensively studied by rhizo team and many molecular tools have been developed to monitoring the nodulation process, including genetic transformation of plants and functional analysis of symbiotic genes based on RNA interference (Gherbi 2008). The use of these molecular biology techniques in the system of D. trinervis-Frankia BCU110501 allow the study of actinorhizal symbiosis within the intercellular infection pathway, and that would lead to comparisons between the two mechanisms involved in the nodulation of actinorhizal plants. The knowledge from such studies would be valuable to better understand the process that leads to actinorhizal symbiosis. Besides that the relevance of studying Discaria trinervis lies in the recent publications supporting the idea of an ancestral mechanism involved in the intercellular infection pathway. Therefore the mechanisms involved in this route of infection could be the key to understanding the evolution of nitrogen-fixing symbiosis, and elucidate the origins of this association between plants and bacteria
- To develop an efficient transformation system allowing the obtention of transgenic roots in Discaria trinervis. This system must also consider that these genetically modified roots do not present any disorder during the nodulation compared to the wild type roots.
- To study in the transformed Discaria trinervis roots the activation of promoters of genes involved in the nodulation process. In a first stage using constructions from heterologous systems in which the genes were previously studied (Casuarina glauca, intracellular infection), to then isolate and study the own genes from D. trinervis. Within the genes to study are CgAux1 (Péret 2007), Cg12 (Svistoonoff 2003), MtEnod11 (Journet et al., 2001) and CgNIN, the orthologue of LjNIN (Schauser et al., 1999), expressed in the early stages of nodulation in C. gluaca and M. truncatula.
- To analyze the expression of the mentioned genes after the inoculation with different strains of Frankia, infective or not, and even using other actinomycetes and rhizospheric bacteria.
- To analyze the ability of different fractions of culture supernatants of Frankia to induce these genes or to interfere with the induction of the same, knowing that these fractions are capable of modifying the kinetics of infection and nodulation in Discaria trinervis.
- To characterize the early responses in Discaria trinervis epidermal and cortical cells during the nodulation process. Functional analysis of the Discaria trinervis Nod factor receptor 5 (NFR5/NFP) and activation of Calcium spiking response to Frankia BCU.
Ø The process of infection through intercellular pathway has mechanisms of interaction and recognition between partners which are partially common and partially different from those presents in the intracellular infection initiated by root hairs deformation.
Ø The differences in both mechanisms of infection result in a differential expression of orthologous genes, but also in the presence or absence of specific genes in one or the other pathway.
Ø Diffusible factors that influence nodulation in Discaria trinervis act by modifying the expression of some of these early interaction genes.
Ø Early stages of the nodulation process, specially those before the intracellular accommodation of the bacteria, differs between Discaria trinervis intercellular infection pathway and the intracellular root hair infection in Casuarina glauca and this also corresponds to differences in gene expression and intracellular dynamics in the host cells.
Development of a transformation system in Discaria trinervis using Agrobacterium rhizogenes
In order to optimize the genetic transformation of Discaria trinervis roots, many parameters involved in the transformations efficiency were tested.
The first step was to test the A. rhizogenes strains. For this purpose plants were transformed with the A. rhizogenes strains A4RS and ARqua1, less virulent, carrying the vector pHKN29 containing the green fluorescent protein gene (GFP) under the 35S constitutive promoter, allowing the detection of transgenic roots under an UV lamp. The transformation with strain A4RS was more efficient, with almost the double of plants expressing GFP compared to the ones obtained with strain Arqua1. However, the morphology of plants obtained with strain A4RS, especially their roots, were markedly different compared to wt plants, therefore it was decided to continue only with the strain Arqua1.
Another parameter to test was the transformation method: in-vitro vs. ex-vitro. The first one requires an axenic handling of the materials, minimizing any potential source of contamination, since plants are cultivate in petri dishes containing full nutrient medium. On the other hand, the ex-vitro transformation does not demand so much carefulness in matters of sterility, and plants are cultivate hydroponically in the greenhouse, with more comfortable handling and reducing costs. Ex-vitro transformation procedure involves stab inoculation of young stalk, with one or two internodes, cut from adult plants cultivated under greenhouse conditions (Collier, 2005). In the in-vitro method A. rhizogenes was inoculated onto D. trinervis seedling by stab inoculation of the hypocotyl or by the passage of sectioned seedling radicles through A. rhizogenes plates.
After five weeks, the percentage of transformed plants expressing GFP in their roots was over 70% by making use of the ex-vitro method and the vegetal material loss was below the 1%. By this time with the in-vitro transformation method, the percentage of lost plants was above the 80% and the number of plants with transgenic roots was below the 10%. The seedling plants were not able to recover after the transformation in the solid culture medium, and there were many of them in which contamination, especially fungi, caused their lost.
According to these results, the ex-vitro transformation method with the A. rhizogenes strain ARqua1 could be a valuable tool for obtaining composite plants with transgenic roots, which would allow the study of actinorhizal symbiosis in D. trinervis at molecular biology levels. For the rest of the experiments the ex-vitro transformation protocol was followed to obtain composite plants with transgenic roots.
Functionality of transgenic nodules
To determine whether genetic transformation of roots somewhat affected nodulation on plants of D. trinervis were carried out observations of histological sections of nodules expressing GFP, as physiological tests and measurement of ARA (Acetylene Reduction Assay). The observations showed similar morphology and anatomy of the nodules of transgenic plants compare to wild type, with numerous infected cells filled of vesicles, where nitrogen fixation could be taking place. The measurement of ARA would finally confirm if nodules from transformed root are fully active and subjected to similar regulation mechanisms. The fact that nodule development stop in inoculated roots if they were started to be watered with N in the watering solution, suggest that main regulation of nodulation is operating in transgenic roots. To complement this observation, GFP transformed roots with nodules will be watered in medium supplemented with nitrogen, to determine whether these nodules are functionally regulated by N in a similar way compare to wild type plants (in progress).
Study of promoters ProCgAux1, ProCg12 y ProCgNIN
To study the activation of the promoters of CgAux1, CG12 and CgNIN in the context of Discaria trinervis, plants were transformed with transcriptional fusions containing the GUS reporter gene linked to the promoter sequences above. The construction with the CgNIN promoter contained the GFP gene directed by the 35S promoter, so that transformed plants could be identified by fluorescence with a UV lamp. The other constructions lacked of this selection marker, therefore transformed roots were indistinguishable from wild type, so it was necessary to use a larger number of plants to ensure that some of them have incorporated the promoter-GUS fusion.
To study the expression of Cg12 and CgNIN promoters, spot inoculation assays were performed with Frankia BCU. In these assays the transformed plants were transferred to growth pouches and then inoculated with Frankia BCU contained in strips of solidified BD -N medium. Frankia strips were placed onto the roots interspersed with control strips without bacteria. Each plant in a pouch had its roots covered by four strips, two with Frankia and two controls without bacteria. Spot inoculation was also tested with the Frankia strains CcI3 and AcN14a non-infective in Discaria trinervis, and the actinomycete Streptomyces coelicolor (Promoter CgNIN).
Finally the response of the promoters of Cg12 and CgNIN was analyzed in the presence of diffusible signal factors (DSF) of Frankia BCU110501. Frankia inoculum was placed in dialysis cellulose tubing (12-kDa cutoff) and mounted on the roots of plants in pouches.
In the assay with the construction ProCgNIN::GUS, about 63% of the A. rhizogenes inoculated plants had roots expressing GFP after 6 week. CgNIN promoter was expressed strongly in lateral root primordium, as well as in root tips prior to inoculation with Frankia. After inoculation, the expression of the promoter was observed in the pericycle, not continuously but in a slot, and in nodules primordium. Expression was also found in epidermal cells, but these observations should be repeated in new plants with better controls, since this type of expression was found in some of the negative controls.
There was no GUS expression in plants transformed with the promoter of Cg12 before inoculation with Frankia. After inoculation with Frankia, GUS expression was only detected in roots that were in contact with Frankia strips. There was no expression of the promoter in the portion of roots in contact with the control strips (without inoculum) in the same plant. Cg12 promoter activity also was not observed when roots were treated with Frankia DSF. Expression of the Cg12 promoter in the roots in contact with the Frankia strips was detected in nodule primordium, particularly in the infection zone (where Frankia invade nodule cells) and in the cells of cortex and epidermis close to the primordium. Expression of GUS in cells of the epidermis took place in areas where the root was in contact with the filaments of Frankia, and that expression extends through the cortex cells to the nodule primordium. In the primordium, the expression is given initially in the region in contact with the cells of the root cortex, by where Frankia penetrates in between the cells, then spread throughout the mature nodule. Expression in nodules is particularly strong in the infection zone, where Frankia becomes intracellular. This may be due to a higher number of cells expressing GUS, which intensify the observed signal, or a change in the expression pattern in which the presence of the bacteria inside the plant cell up regulate the expression from the promoter.
In the case of CgAux1 promoter, reporter gene expression in uninoculated roots was observed in the vasculature and in the apical meristems of lateral roots. Expression of the promoter in the roots inoculated with Frankia follows the same pattern as in uninoculated roots, even in nodules, where GUS expression was observed in the vascular bundles and at the apex, but not in Frankia infected cells. There are not infection-related activation of the CgAux1 promoter in Discaria trinervis transformed roots and this differs from the expression pattern observed in C. glauca. This results were really interesting and stimulates us to go deeper in the understanding of auxins behaviors in Discaria trinervis nodules. For this propose we transformed Discaria trinervis plants with a construction carrying the DR5 auxin response element fused to the VENUS reporter gene. DR5 provides a useful reporter system for studying auxin-responsive transcription in the root tissues (Ulmasov et al. 1997). In non-inoculated plants, the reporter gene is expressed in the apical meristem of the roots and roots primordium. In plants inoculated with Frankia BCU the expression patterns of the VENUS protein is similar to that observed in the primordium of lateral roots during the early stage of the nodulation. We are currentlyconducting some experiments to observer the expression pattern of DR5 in full development nodules and to test if this reporter system actually responses to auxins concentration in Discaria trinervis root by applying different auxins to the transformed root.
In parallel we made immunohistochemistry analysis using fluorescent labeled antibodies to detect the auxin PAA (Phenyl Acetic Acid) and the auxin efflux transporter PIN1. The results showed that the PAA accumulation pattern is conserved between Discaria trinervis and Casuarina glauca nodules (Perrine-Walker et al. 2010), but the distribution of PIN1 transporter differs among the two actinorhizal plants. Experiments in which wild type Discaria trinervis plants were treated with NOA (naphthoxyacetic acid, an auxin influx inhibitor) showed that the nodulation is affected by the treatment with this auxin transporter inhibitor, a result similar with those observed in Casurina glauca (Péret et al. 2007). In summary this seems to indicate that auxins are involved in the nodulation process in Discaria trineris as it known in Casuarina glauca, but the mechanism of accumulation of the hormone within the symbiotic tissue would be different between the two systems.
Discaria trinervis Dt12 and DtAux1 sequence isolation and expression
To determine if the results obtained with heterologous promoters reflect the behavior of the endogenous genes from D. trinervis, the full sequences of the genes corresponding to Cg12 (= Dt12) and CgAux1 (=DtAux1) were isolated with a Genome Walking approach. The alignment of these sequences with the corresponding C. glauca genes revealed 90% of identity at aminoacid level between DtAux1 and CgAux1, and 65% of identity between Dt12 and Cg12. The expression of Dt12 and DtAux1 was then analyzed on roots and nodules by a quantitative RT-PCR approach. Results showed that Dt12 is highly expressed in nodules but not in roots. On the other hand DtAux1 is expressed in both organs, but twice as many transcripts are found in nodules compared to roots. These results suggest that the endogenous D. trinervis genes are activated in a similar way as the ones from C. glauca.
A Dt12/DtAux1 promoter GUS/GFP reporter constructs were made by a Golden Gate approach using Type II restriction enzymes. We began to test the expression pattern of the endogenous Dt12 promoter by GUS staining and confocal fluorescent microscopy after the inoculation with Frankia BCU. The behavior of the Discaria endogenous Dt12 promoter is similar to that observed with the Casuarina glauca promoter. Interestingly we could not observe the expression of promoter Dt12 in individual epidermal cells. The reporter gene expression always comprise at least two adjacent cells. The bacteria are necessary surrounding by at least 2 host cells while steel intercellular, and that explain our observations. This supports the idea of the Dt12 promoter as a marker of the intercellular infection in Discaria trinervis – Frankia BCU system, when the activation of the promoter take place in the root cells surrounding the growing bacteria that moves throughout the root tissue. We have samples of transgenic root transformed with the ProDt12::GUS construction to look at the electronic microscopy to confirm the intercellular localization of the Frankia and to see to what extent the expression of Dt12 promoter in the host cells is associated with the penetration of bacteria inside the root tissue.
Finally we are doing functional genomic in Dt12 gene by the use of an RNAi silencing approach. For that propose we made 2 RNAi constructions, one directed to the sequence corresponding to the active site of the protein, and a second one directed to the 5 'UTR of the mRNA. This experiment is now in progress and we expect to analyses the results in the following months, closing the story with the Dt12 gene. A similar study is currently being performed in the group witrh the C. glauca gene, Cg12 (Cg12RNAi).
The DtAux1 promoter constructions were more difficult to make because of some problems in the isolation of the promoter sequence. These constructions are now completed and will be tested in the next months, which added to the results with DR5, NOA (auxin transport inhibitor) and the PIN1/PAA antibodies will mark the end of the work with DtAux1 gene.
NOD signaling pathway in Discaria trinervis
The last chapters of this thesis will focus on the early recognition stages between the host plant and the symbiotic bacteria. We want to study host signaling process and cellular reprogramming prior to and during intercellular Frankia infection in Discaria trinervis and compare this results with the same experiments in Casuarina glauca intracellular infection. We are particularly interesting in the signaling process involve in the recognition of the bacteria by the host plant. In legumes the early recognition of the symbiont by host plant takes place through the Nod Factor Receptor (NFR1/LYK3 & NFR5/NFP) that specifically perceive the lipochitooligosaccharides (Nod Factors) synthesized by the bacteria. Because of this we are interesting in understand the role of this receptors in Discaria trinervis and to know whether the NOD factor signaling pathway is conserved in this actinorhizal plant.
We isolated the complete sequences of the homologous gene of NFR5/NFP from legumes in Discaria trinervis. We made an RNAi construct directed to the sequence corresponding to the kinase domain of the protein (as in Rik Op den Camp et al. 2010). A preliminary result shows no differences in the nodulation between RNAi plants and controls.
We also transformed Discaria trinervis with an auto-active version of the Casuarina glauca CCamK (Calcium Calmodulin Kinase) gene that induces spontaneous nodulation in Casuarina glauca. This gene also induces spontaneous nodulation in Discaria trinervis which demonstrate that the NOD signaling pathway is also conserved in this actinorhizal plant. Farther we will study the process upstream the CCamK gene, particularly what happens in relation to the Calcium spiking and the cellular dynamics in the cells surrounding the bacteria during the intercellular infection. Our approach involves the use of a nuclear-targeted camaleon reporter (Sieberer et al. 2009) to follows the intranuclear calcium oscillations in response to Frankia inoculation and an endoplasmic reticulum-targeted mCherry reporter gene to observed changes in the cellular dynamics during Frankia infection.
- Inoculation of Discaria trinervis ProDt12::GFP transformed plants with Frankia strains belonging to the Eleagnus group which are known to develop nitrogen fixing nodules in a wide range of host plans, and also with some strains which have the ability to infect the root tissue but do not develop nitrogen fixation nodules. The idea is to test whether the Dt12 promoter respond to the infection by a heterologous Frankia strain and to see if this activation is specific to symbiotic bacteria infection process or if is generalized to infection process taking place by any bacteria.
- Localization of Frankia BCU in the root and nodule tissue using histochemistry approach with fluorescent labeled WGA (Wheat Germ Agglutinin) and GUS staining to confirm the promoter activity of Dt12 as a marker of intercellular infection process (fluorescent confocal microscopy and electron microscopy).
- Functional analysis of the Dt12 gene by RNAi silencing approach.
- Confirm the ProCgAux1::GUS activation pattern with the endogenous DtAux1 promoter constructions.
- Calcium spiking and cellular dynamics associated with Frankia infection through nuclear camaleon and endoplasmic reticulum-targeted mCherry reporter genes (FRET based Fluorescent Confocal microscopy).
The firstthesis committeewas held atIRDresearch center in Montpellier onJune 23th 2010with the presenceof the juries Dr. FabienneCartieaux (UMR LSTM, IRD,
A second thesis committee will be held in Argentina in late September with the presence of the following jury: Dr. Claudio Valverde (PIB, Universidad Nacional de Quilmes, Bueos Aires, Argentine), Fernando Bravo (Universidad Nacional de Buenos Aires,
Finally we propose a possible date for the final defense of the PhD thesis by the end of September 2013.
" Testing the antifungal activity and phytochemical analysis of extracts of local plants of Burkina Faso for the control of groundnut (Arachis hypogaea L.) foliar diseases".
- PhD thesis staff: Pr. Philippe Sankara, Claudine Campa, Yves Baissac
- Date of PhD thesis : July 2013
This work, under the direction of Professor Philippe Sankara, proposed to study the antifungal properties of some aqueous extracts of local plants to control foliar diseases of peanut. An in vitro evaluation has highlighted the antifungal efficacy of the extracts of Lippia multiflora Moldenke, Boscia senegalensis (Pers.) Lam, Ziziphus mucronata Willd., Azadirachta indica A. Juice. and Securidaca longepedonculata L.. These plant extracts were tested on in vitro cultures of spores from Cercospora arachidicola Hori., Phaeoisariopsis personata (Berk. and Curt.) and Puccinia arachidis Speg., fungi that are agents of early leaf spot, late leaf spot and rust for Arachis, respectively. An inhibition rate of germination was noticed for all the spores, from 38 to 100% for Cercospora spores and from 4 to 71 % for urediniospores of rust.
Field trials were conducted from 2009 to 2011 in two sites differing by the environmental conditions (Gampela and Niangoloko). In both sites, three Arachis varieties, more or less sensitive to diseases, were used to assess the impact of treatment of aqueous plant extracts on the development of the disease, defoliation and on yield components. A substantial increase in pod yield was observed when plots were treated with aqueous plant extracts, especially with Lippia multiflora for the sensitive variety TS31 -1.
A phytochemical analysis carried out at the IRD in Montpellier (SMART lab, DIADE) on Lippia multiflora and Ziziphus mucronata extracts allowed identifying several antifunga molecules such as verbascoside, samioside , quercetin and rutin.
" Hormonal control of lateral root development in Arabidopsis thaliana "
- PhD thesis staff: Laurent Laplaze, Malcolm Bennett (UoN), Soazig Guyomarc'h
- Date of PhD thesis: October 2013
The ability of plants to exploit the ressources from the soil is highly dependent on their root architecture. It is determined by both root growth and root branching i.e. by de novo organogenesis of lateral roots (LR). Over the last decades genetic approaches enabled ones to identify crucial components of the gene regulatory networks (GRN) controlling LR formation. However these approaches are unable to rapidly identify large-scale GRN. A system biology approach was undertaken to unravel the GRN controlling LR initiation and patterning in Arabidopsis . This approach consists in infering the structure of the GRN from a time-series transcriptomic dataset generated in Nottingham and subsequently in experimentally validating the predictions. A novel reverse-engineering algorithm was developed and used on a subset of 80 genes known to be involved in root development. Most of the known interactions were sucessfully recovered by the algorithm and the overall structure of the predicted network makes sense with the currently available experimental data. The predictions concerning the targets of two transcription factors known to play a crucial role in LR development were tested by making use of an additional transcriptomic dataset (Fukaki et al., personal communication). This showed that the algorithm has a very good sensitivity (>50%) and an excellent specificity (>98%) for at least those two nodes. The goal is now to validate a few other potentially meaningful predicted interactions. The spatio-temporal expression pattern of the genes involved in those interactions will be examined in detail in wild type and mutant backgrounds. Furthermore the directness of the interactions will be tested by ChIP-PCR.
"Search for genes regulated by CRL1, a transcription factor controlling the development of adventitious roots in rice, and study their genetic diversity."
- PhD thesis staff: Pascal Gantet
- Date of PhD thesis : April 2013
Rice is the first cereal for human consumption. It is consumed by more than half the world's population (Khush, 2005). Among four types of rice, it has the largest irrigated area (53%) and has the best performance (75% of world production). However, this type of rice is very greedy water: it takes about 5 tons of water to produce 1 kg of rice.
Because of population growth, pollution of the environment, the development of rice is now limited by the decrease in the area of culture, soil contamination by salt due to rising water sea in the deltas and limited water resources. It then became necessary to develop rice varieties with high yield but also tolerant to water stress to maintain a good level of production with less water available.
For this purpose, it is necessary to improve the development of the root system so the plant can make better use of water resources in the ground and avoid water stress. Deeper roots and branched allow the plant better access to water in the soil. In order to identify genetic determinants of the development of the root system, we chose to study regulatory networks of genes that control root development.
Research has shown that CRL1 is a transcription factor, controlling most upstream gene regulatory network that controls the development of adventitious roots in rice.
My thesis topic is to search for genes regulated by CRL1 and study their correlation with nucleotide diversity in root development in a collection of rice varieties Vietnamiene.
" Leaf fiber of Astrocaryum chambira Burret (Arecaceae) and related species, histological and physicochemical characterization"
- PhD thesis staff: Francis Kahn
- Date of PhD thesis : December 2012
Anatomy as well as physical and chemical properties of the commercial leaf fiber of Astrocaryum chambira are determined and compared with other species of the genus (Astrocaryum jauari, A. standleyanum, A. perangustatum) in order to understand why this fiber is so commonly extracted and used in the western Amazon, region of distribution of this species. Chambira fiber consists of non-vascular fiber strands closely adhered to the adaxial hypodermis. Non-vascular fibers are very long and have a high content of cellulose while the ultrastructure of their cell wall reveals that it consists of three sub layers. Thickness and tensile strength are higher in chambira fiber than in others species. As a result, chambira fiber is comparable by its great length and resistance to other vegetal fibers used by the industry
"Cryopreservation and cryotherapy of grapevine (Vitis vinifera L.)"
- PhD thesis staff: Florent Engelmann and Jasminka Kontic (Univ. Zagreb, Croatia), France/Croatia supervision
- Date of PhD thesis defense: December 2013
Grapevine (Vitis L.) is an economically important crop for Croatia. The search for virus diseases in different vine-growing regions of Croatia has revealed the widespread presence of viruses and the low occurrence of non-infected vines. For some of the native cultivars and often neglected ones there not a single healthy plant.
Cryopreservation, the storage of biological material at ultra-low temperature (liquid nitrogen, -196ºC) is considered an ideal means for long-term conservation of germplasm. All cellular divisions and metabolic processes stop. The plant material can thus be stored for extended time spans without any change.
Recently, it has been shown that cryopreservation can successfully eliminate viruses from shoot tips through a process termed cryotherapy. Cryotherapy is based on a cellular selection occurring during cryopreservation, the meristematic cells, which are devoid of vituses, withstanding liquid nitrogen exposure, while more differentiated cells, which contain viruses are killed by cryopreservation. Cryotherapy has been efficiently performed on grapevine to eliminate Grapevine virus A (GVA).
Cryopreservation hence appears as an efficient mean for virus eradication through cryotherapy and for long-term preservation of valuable grapevine cultivars.
The PhD thesis project of Zvjezdana Marković includes three main topics:
- Optimize the cryopreservation procedure for shoot tips of autochtonous grapevine cultivars. Different cryopreservation techniques (encapsulation-dehydration, vitrification, encapsulation-vitrification and droplet-vitrification) will be tested and compared for their efficiency and applicability. Different steps and parameters of the protocols will be studied in order to optimize the percentage of survival and regrowth of shoot tips. Following an initial training period in Montpellier, this research topic will be implemented in Croatia.
- Compare the efficiency of different techniques for virus eradication. The classical meristem culture technique, in combination, or not, with thermotherapy and cryotherapy in combination, or not, with thermotherapy, will be compared for their efficiency to eliminate selected grapevine viruses. ELISA and PCR techniques will be used to detect viruses. This topic will be carried out in Croatia.
- Study the localisation of viruses in shoot tips, at different steps of the cryopreservation protocol, using immunolocalisation techniques.This part of work will be performed in Montpellier, trough a collaboration between IRD and CIRAD.
The above studies will produce original results on: 1) the influence of different parameters of the cryopreservation protocol on shoot tip structure integrity; 2) the efficiency of different virus eradication techniques (meristem culture, thermotherapy, cryotherapy); and 3) the localisation of viruses in grapevine shoot tips after different treatments usingo immunolocalisation. From an applied perspective, this research will lead to the production of virus-free grapevine cultivars, which can be used in the nursery and in the genebank to establish a collection of Croatian native cultivars.
"Structure of nuclear and chloroplastic genetic diversity in Algerian date palm"
- PhD thesis staff: Nadia Boughedoura et Jean-Christophe Pintaud
- Date of PhD thesis defense: 2013
Algeria possesses a rich date palm diversity, widely distributed in the submediterranean and Saharian regions of the country. Although a thousand of date palm cultivars have been reported for the whole country, very few (like Thorry abd Deglet Nour) have been studied genetically. For this study, a sampling of 450 individuals of Phoenix dactylifera representing 150 varieties from 11 oases in eastern, central and western Algeria, have been constituted. This material is genotyped with 18 nuclear SSR markers and two chloroplast markers (a minisatellite and a SNP). Cultivar diversity and geographic structure are studied, and a genetic identification key is built using 7 markers. in addition, a battery of chloroplast loci is sequenced in 12 varieties having the so-called Oriental chlorotype (from Asia) and 12 with the so-called Occidental chlorotype (from Africa), in order to characterize the molecular divergence between these two genomes.
"Phylogeny of subtribe Mauritiinae and variability of Mauritia flexuosa
- PhD thesis staff: Rina Ramirez. et Jean-Christophe Pintaud
- Date of PhD thesis defense: 2013
The subtribe Mauritiinae, composed of 3 genera and 7 species, is the only Neotropical group in subfamily Calamoideae, that contains in particular the African and Asian rattans. In contrast to the rattans, which are climbing and have pinnnate leaves, Mauritiinae are arborescent with palmate leaves. A fruit crop specie, Mauritia flexuosa, is currently under domestication. Although it is a numerically small group, Mauritiinae are highly diversified on morphological and ecological grounds. In this thesis, a complete species-level phylogeny of Mauritiinae is constructed using chloroplast sequences, and the genetic diversity of Mauritia flexuosa in Peru is investigated using ISSR and SSR markers.
" Characterization, productivity, and trade network of the fruit of the palm “aguaje” (Mauritia flexuosa L.f.) in the provinces of Rioja, Moyobamba, San Martín, Mariscal Cáceres and Lamas (San Martín region, Peru)"
- PhD thesis staff: Francis Kahn
- Date of PhD thesis : December 2012
In the region San Martín harvesting fruit of the palm Mauritia flexuosa L.f. (aguaje) is no more a predatory activity by killing the plant. Several systems are currently used for climbing and collect the fruits. A first part of the study deals with fruit characterization and productivity. Aguaje palms produce an average annual mean of 4.6 bunches per individual. The plant start fructifying at 6m in height in natural vegetation and at 3-4m in cultivated area. Each bunch bears a mean of 583 fruits; a palm produces an annual mean of 2650 fruits (ca. 1.4 ton). Fruit is scaly, oval, 4.9 cm long, 4.1 cm in diameter, with a mean weight of 56.6g (edible mesocarp 30%, endocarp 60%, epicarp 20%). The fruit is sold in rural and urban regional markets in the Peruvian Amazon and constitutes a significant source of income. The second part of the study analyzes the trade network. It is a complex chain that includes many actors, as producers and operators, intermediaries, conveyors, retailers and consumers. The important points of marketing are located in the main urban centers of the San Martín region (Tarapoto, Moyobamba y Rioja). Fruits sold in these cities also come from areas located in the eastern Amazonian lowlands (Loreto region).
Morphological, genetic and cytogenetic diversity of Mascarocoffea (Coffees from Indian Ocean islands). Evidence of non African origin of coffees."
- PhD thesis staff: Perla Hamon and Jean-Jacques Rakotomalala
- Date of PhD thesis : May 2012
The new classification and repartition of the genus Coffea (124 species including ex-genus Psilanthus native to Africa, Indian Ocean islands (IOIs), India, southeast Asia and Australasia) rises the question on its origin. Genetic and cytogenetic data are available for African species but not for Mascarocoffea (coffees from IOIs including half part of the species). The aims of this thesis were i) to estimate the morphologic, genetic (13 SSRs) and cytogenetic divergence of and among Mascarocoffea; ii) to produce a molecular phylogeny and iii) to reconstruct the coffees evolutionary history.
Although the global morphological diversity appears as a continuum, it permits the identification of groupings and obvious specific reproductive strategies. Genomes size varied from 0.96 to 1.41 pg/2C with an increasing global trend from north to southeast Madagascar.
Average alleles number per locus is high whatever the region and 20% total alleles are shared by African and IOIs coffees.
The molecular phylogeny shows a basal position for Psilanthus, clear divergence between African and IOIs species. Clearly, IOIs clade is not nested within the African. Our results and information from other works permit us to elaborate a new scenario for the evolutionary history of coffees. The centre of origin should be located in India. Then two primary centres of diversification occurred; one in Kenya- Ethiopia, Africa the other in north Madagascar. From these centres, migrations towards centre and west and towards east Africa led to secondary centres of diversification while in Madagascar, migrations towards southeast and towards the other islands led to radiative speciation.
Keywords : Coffea, Mascarocoffea, Psilanthus, Madagascar, Africa, COSII, molecular phylogeny, SSR, flow cytometry, phenotypic diversity.
“Ecological and ecolutionary studies in the wax palm genus Ceroxylon (Areceacea: Ceroxyloideae) with emphasis on C. quindiuense”
- PhD thesis staff: Gloria Galeano (Instituto de Ciencias Naturales, Universidad Nacional de Colombia), Jean-Christophe Pintaud
- Date of PhD thesis : March 2013
The fragmentation of plant communities is hapless because it diminishes resilience through, among other factors, diversity loss and genetic impoverishment of populations caused by the obstruction of gene flow, especially along wide distribution ranges. Fragmentation also affects population structure, escalating genetic drift, and irreversibly accelerating genetic erosion, via the alteration of the ecological population structure. The long-term effect of these phenomena is the loss of response capacity (resilience) against the variable environmental scenarios, thus an increased vulnerability to extinction. The Andean Palms of genus Ceroxylon are ecologically very important as some can form large and dense stands, supply long-year food for many species of birds and mammals, offer nesting sites for a wide variety of Psittacids, and display complex pollination syndromes. Nevertheless, all of the species are to some extent endangered, mainly due to habitat loss entailed in unsustainable agricultural practices, extensive livestock and ranching, and the high-consuming urban lifestyles that, in the Andes, grow by the day. Also, vulnerability to extinction within these species is enhanced by their long life cycles, their dioecious nature, and their habitat specificity. In midst of the environmental emergency that the Andes face today, Ceroxylon palms represent highly informative conservation targets, as they are not only clearly conspicuous integrants of Andean plant communities, but possess life traits that favor age, sex and health recognition, and also social acceptance and identification. This project aims at the following: a) Studying how ecosystem fragmentation and landscape discontinuities affect the ecological population structure of C. quindiuense, b) Quantifying and comparing genetic diversity in C. quindiuense between different age classes, c) completing a Phylogenetic and Network analysis of Ceroxylon focusing on the C. quindiuense / C. ventricosum complex (Andes of Colombia, Ecuador and Peru). Ultimately, it aims at providing information (obtained in a-c) that is necessary to envisage in the near future, a conservation plan for the species that can be extended to the whole genus, and strategically to the Andes of Colombia.
"Study of the genetic and phenotypic variability of some important date palm cultivars in Algeria"
- PhD thesis director: Nadia Boughedoura et Jean-Christophe Pintaud
- Date of PhD thesis defense : 2014
In Algeria, clonal multiplication of cultivars is the rule, performed by separation of offshoots from mother plants. This asexual mode of propagation does not prevent all genetic variability due to the existence of somatic mutations. Spontaneous somaclonal variation of date palm cultivars has never been studied, all studies of this kind focussing on conformity of in vitro regenerants to type. Four cultivars having different behaviours have been choosen: Deglet Noor, the elite variety for which a stringent selection of the standard type is expected, leaving few somaclonal variation; Ghars, Mech Degla and Faggous, three varieties of lower quality in which somaclonal variation is supposed to be uncontrolled. Molecular markers used are SSR and ISSR. Phenotypic variation is also studied at the level of fruit and seed variability at different scales (intra-infructescence, intra-tree, inter-tree). Moreover, controlled pollination with different pollens is carried out in order to identify a potential metaxenic effect.
"Inference of demography and selection in plant species"
- PhD thesis director: Martin Lascoux
- Date of PhD thesis : August 2013
The distribution of nucleotide variation along the genome is one of the main sources of information when attempting to reconstruct the evolutionary history of species. Until recently inferences based on nucleotide variation could roughly be divided into two groups. Population geneticists focused on within species variation while systematicists and phylogeneticists focused on the divergence among species. Until recently the two fields evolved to a large extent separately but they have recently started to slowly merge, as it became increasingly obvious that (i) the same processes underly polymorphism and divergence, (ii) many species still share ample levels of polymorphisms and (iii) how much both fields could gain from it. A clear example of this trend is the development of Isolation-with-migration (IM) models. One of the main aims of these models is to estimate how much of the shared polymorphisms between two diverging species are ancestral and how much are due to gene flow after the two species diverged. While the IM models undoubtedly are a step forward they also have obvious limitations. In complex of species considering a pair of species may be somewhat artificial, it is not clear either how much of the within species history can be safely ignored and they may still be too rough to tell apart a progressive divergence with limited gene flow from secondary contact. The development of more general approaches is therefore needed. As polymorphism data is accumulating in many species and as those often show large amount of shared polymorphisms we may also need to rethink the standard neutrality tests and find clever ways to use these new data when trying to disentangle the effects of selection and demography on DNA polymorphisms. For instance, a gene showing a large excess of common variants in species with different histories could be evidence of selection acting on the gene.
The present thesis will focus on polymorphism and divergence in two groups of plant species with very contrasted life-histories. Picea (spruces) is a fairly large genus of trees with long generation time and high level of outcrossing that started diverging some 70 million years ago. In contrast, Capsella is a small genus of three annual plant species (C. grandiflora, C. rubella and C. bursa-pastoris ), two of which are selfers (C. rubella and C. bursa-pastoris ). That started diverging a mere hundred of thousand years ago. In both cases we already have a fairly good amount of data. In Picea one will focus on inferring polymorphisms and divergence in a group of species from the Tibetan Plateau and on the analysis across species of specific genes that showed evidence of selection in genome scans in Picea abies. In Capsella the initial focus will be on the joint analysis, using Approximate Bayesian Computation, of the Isolation-with-Migration of the three species. We recently showed in pairwise analyses that there had been gene flow between the two selfers and that C. rubella derived from C. grandiflora as recently as 20,000 years ago."
"Functional diversity of genes involved in rice panicle architecture."
- PhD thesis staff: Pascal Gantet, Stéphane Jouannic and Hélène Adam
- Date of PhD thesis defense: Octobre 2014
Inflorescence structure is a key character in the process of domestication, which often involves a change in the pattern and timing of branching, and ultimately the yield, since it directly affects the numbers of flowers and seeds produced. An understanding of the molecular mechanisms involved in panicle development in Asian and African rice species is an important issue for yield improvement breeding programs, through the identification of functional nucleotide polymorphisms (FNPs) suitable for marker-assisted selection (MAS). Cellular events associated to panicle development and genes affecting the panicle architecture and the seed number per panicle have already been described in O. sativa. Nevertheless, FNPs associated to these genes in O. sativa and/or other AA genome rice species have not been reported. Moreover, little is known about developmental and molecular mechanisms associated to panicle architecture in African rice.
The main objective of this PhD project is to identify the determinants of the panicle architecture and function under optimal conditions and to assess their allelic diversity in both Asian and African species (cultivated and wild-relative species). The first part of the project will concern the analysis of the inter-specific diversity between cultivated and wild-relatives in Asia and Africa. The second part of the project will be specifically dedicated to the analysis of the panicle diversity of Vietnamese accessions.
"Diversity of the genes of resistance against Rice yellow mottle virus (RYMV) in African cultivated rice, Oryza glaberrima Steud: interest for the development of a durable resistance."
- PhD thesis staff: Laurence Albar
- Date of PhD thesis : October 2012
Rice yellow mottle virus (RYMV) is one of the main pathogens of rice in Africa. Only two accessions of the Asian cultivated rice species, Oryza sativa are highly resistant. This resistance is controlled by the RYMV1 gene, coding the translation initiation factor, eIF(iso)4G. A single résistance allele has been identified in O. sativa. Heavy crop losses due to the disease and the rarity of high resistance in Asian cultivated rice are a constraint that justifies the characterization of new sources of resistance in African cultivated rice, O. glaberrima. The screening of RYMV resistance of 337 accessions representative of the geographical and agro-ecological diversity of the African rice species O. glaberrima and genetic basis of high resistance analyses were performed. A partial resistance was observed, but, appeared to be independent of morphological traits, contrary to what is known in O. sativa. Furthermore, 29 highly resistant accessions were identified. Analysis of the allelic diversity of the RYMV1 gene in these accessions and genetic complementation assays has allowed the identification of a third resistance allele of RYMV1, rymv1-5, and two new resistance genes RYMV2 and RYMV3. O. glaberrima thus constitutes a major source of high resistance against RYMV, with both a higher frequency and a greater diversity of resistance than in O. sativa. The RYMV2 gene was mapped on rice chromosome 1 in a 0.4 Mb interval in the Nipponbare variety and its positional cloning is in progress. Molecular markers specific to the different alleles of the RYMV1 gene were developed for marker-assisted selection of resistant genotypes for disease management. Furthermore, the resistance breaking of RYMV1 and RYMV2 resistance alleles, individually or in combination, was assessed. The rymv1-5 resistance allele and allelic combinations such as rymv1-3/rymv2-2 and rymv1-5/rymv2-2 were particularly difficult to overcome and represent excellent candidates genes to improve the resistance to RYMV in Africa.
Keywords: Oryza glaberrima, Rice yellow mottle virus, Resistance genes, allelic diversity