domain: sdv sde sdv.bv

text:
Svenja Heesch

Ga Youn Cho

Akira F Peters

Gildas Le Corguillé

Cyril Falentin

Gilles Boutet

Solène Coëdel

Claire Jubin

Gaelle Samson

Erwan Corre

Susana M Coelho

J Mark Cock

A sequence-tagged genetic map for the brown alga Ectocarpus siliculosus provides large-scale assembly of the genome sequence

Keywords: chromosomes, Ectocarpus siliculosus, genetic linkage map, microsatellites, model organism, Phaeophyceae

 Ectocarpus siliculosus has been proposed as a genetic and genomic model for the brown algae and the 214 Mbp genome of this organism has been sequenced. The aim of this project was to obtain a chromosome-scale view of the genome by constructing a genetic map using microsatellite markers that were designed based on the sequence supercontigs.  To map genetic markers, a segregating F 2 population was generated from a cross between the sequenced strain (Ec 32) and a compatible strain from northern Chile. AFLP analysis indicated a significant level of polymorphism (41%) between the genomes of these two parental strains. Of 1,152 microsatellite markers that were selected for analysis based on their location on long supercontigs, their potential as markers and their predicted ability to amplify a single genomic locus, 407 were found to be polymorphic.  A genetic map was constructed using 406 markers, resulting in 34 linkage groups. The 406 markers anchor 325 of the longest supercontigs onto the map, representing 70.1% of the genome sequence.

 The Ectocarpus genetic map described here not only provides a large-scale assembly of the genome sequence, but also represents an important tool for future genetic analysis using this organism.

Introduction

Brown algae are an ecologically and economically important group of organisms. They are the dominant organisms in most coastal ecosystems, providing habitats for a wide range of other species, and are exploited both as a food source and for biomolecules such as alginates and fucans. As our understanding of these organisms improves, additional biomolecules of interest are being developed, such as the use of the brown algal storage polysaccharide, laminarin, as an elicitor of defence responses in crop plants [START_REF] Klarzynski | Linear -1,3 glucans are elicitors of defense responses in tobacco[END_REF]. Brown algae are also one of the groups that have been put forward as having a high potential as a source of biofuel (reviewed in [START_REF] Bartsch | The genus Laminaria sensu lato: recent insights and developments[END_REF].

The brown algae are members of the stramenopiles and, as such, are distantly related to other major eukaryotic groups such as the animals and fungi (opisthokonts) or land plants and green algae (plantae). As a result of this phylogenetic position, the brown algae exhibit many novel features compared with these other groups, and this is one of the reasons why these organisms are of interest for the development of novel biomolecules. Another consequence of the large phylogenetic distance that separates brown algae from the plantae and the opisthokonts is that model organisms from the latter groups are only of limited relevance to understanding brown algal biology. In order to study the novel features exhibited by this group at a molecular level, it is therefore necessary to develop model organisms that are better adapted to studying brown algal biology.

Over the last two centuries, Ectocarpus siliculosus has been used as a model organism to study many aspects of algal biology [START_REF] Charrier | Development and physiology of the brown alga Ectocarpus siliculosus: two centuries of research[END_REF] and several groups are currently focusing their effort on the development of modern genetic and genomic tools for this organism (Peters et al., 2004a;[START_REF] Coelho | Complex life cycles of multicellular eukaryotes: new approaches based on the use of model organisms[END_REF]. This recent work has involved the sequencing of the complete 214 Mbp genome of this alga (http://www.cns.fr/spip/-Ectocarpus-siliculosus-.html, Cock et al., submitted) and the development of a range of molecular, genomic and genetic tools including large collections of cDNA and small RNA sequences, whole genome tiling microarrays, EST-based expression microarrays and mutant screens [START_REF] Peters | Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus[END_REF][START_REF] Dittami | Global expression analysis of the brown alga Ectocarpus siliculosus (Phaeophyceae) reveals large-scale reprogramming of the transcriptome in response to abiotic stress[END_REF]Cock et al., submitted;unpublished data). Ectocarpus was chosen as a model organism because it has several features that make it amenable to genetic approaches. These include its small size, its relatively short life cycle (three months), which can be completed under laboratory conditions, it's high fertility and because controlled genetic crosses can be carried out (Peters et al., 2004a). The haploiddiploid life cycle of Ectocarpus provides a means to directly assess the effects of genetic variations (such as mutations) in a haploid context, without the confounding effects of heterozygosity and dominance. Moreover, all the meiospores in a unilocular sporangium are derived from a single meiotic event (which is followed by several mitotic divisions), allowing analysis of the progeny of a single meiosis [START_REF] Knight | Studies in the Ectocarpaceae. II. The Life-History and Cytology of Ectocarpus siliculosus, Dillw[END_REF][START_REF] Peters | Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus[END_REF].

Here we describe the construction of a sequence-anchored genetic map for Ectocarpus. The genetic map was constructed for two reasons. The first objective was to create a tool for genetic analyses in this species, such as associating phenotypes with genetic loci, mapping genetic mutants and as a starting point for positional cloning projects. The second objective was to create a large-scale assembly of the Ectocarpus genome sequence (a process that is also referred to as super-scaffolding). The genome has been sequenced to 10.5x coverage and the sequence data has been assembled into 1,565 supercontigs (i.e. chains of contiguous contigs separated by short sequence gaps, often also referred to as scaffolds). This assembly represents a high quality draft genome sequence but no information has been available about how these supercontigs are organised at the chromosome scale. In this study we have mapped microsatellite (or simple sequence repeat, SSR) markers derived from 325 of the longest supercontigs to anchor these sequences onto a genetic map. This has allowed 137 Mbp (70.1% of the genome) of DNA sequence to be associated with genetic linkage groups. The genetic map, therefore, both represents an important tool for future genetic analysis in Ectocarpus and provides a large-scale assembly of the genome sequence that will be invaluable for identifying genome-wide trends in Ectocarpus.

Materials and methods

Algal strains, culture conditions and DNA extraction

The segregating mapping population was generated by crossing Ectocarpus siliculosus strain Ec 32 (CCAP 1310/04; [START_REF] Peters | Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus[END_REF] with strain Ec 568 (CCAP 1310/334), isolated from Arica, northern Chile [START_REF] Peters | Diversity of Ectocarpus (Ectocarpales, Phaeophyceae) in Peru and northern Chile, the area of origin of the genome-sequenced strain[END_REF]. Production and raising of zygotes was performed by combining fertile gametophyte thalli of the two parental strains in a hanging drop preparation, as described (Peters et al., 2004b). Each of the 60 individuals of the segregating F 2 population was derived from a different unilocular sporangium (i.e. from separate meiotic events) on the same hybrid F 1 sporophyte (strain Ec 569, CCAP 1310/335). The progeny were maintained as haploid lines by cultivating asexual partheno-sporophyte progeny of the F 2 population of gametophytes. Strains were cultivated at 13°C in white fluorescent light of 10-30 mol m -2 s -1 photon fluence rate and a day length of 10 hours light: 14 hours dark in a walk-in culture room. The culture medium was half-strength, Provasolienriched [START_REF] Starr | UTEX -The culture collection of algae at the University of Texas at Austin 1993 List of cultures[END_REF], autoclaved seawater. DNA was extracted from about 800 mg of tissue that had been frozen at -80°C by grinding the material in liquid nitrogen and extracting using either a commercial kit (Macherey & Nagel, Germany), according to the manufacturer's instructions, or a CTAB extraction protocol (Cock et al., submitted). The DNA was purified on a CsCl gradient using Hoechst bis-benzimide 33258 (Sigma-Aldrich, Buchs, Switzerland) as the fluorescent stain. DNA yield and quality was assessed using a spectrophotometer (Nanodrop, Wilmington, USA) and by agarose gel electrophoresis.

AFLP analysis of polymorphism

AFLP analysis was carried out as described by [START_REF] Vos | AFLP: a new technique for DNA fingerprinting[END_REF]. A total of 250 ng of template DNA was digested with EcoRI and MseI before addition of adapters. In contrast to the original protocol, digested DNA was not diluted ten-fold before the ligation step. The digested and ligated DNA was then used for pre-selective amplification with EcoRI+A (GACTGCGTACCAATTCA) and MseI+C (GATGAGTCCTGAGTAAC) primers. Pre-selective amplification mixtures were diluted 50-fold with TE buffer and then used in selective amplification. Selective amplifications were carried out with +3 nucleotide EcoRI and MseI primers (Supplementary table S1). The EcoRI primers were labelled with a fluorescent dye (IRD 700 or IRD 800). AFLP PCR products were analysed on a Licor LongRead IR4200 DNA analyzer with a 5.5% polyacrylamide gel matrix (LI-COR Biosciences Ltd, Cambridge, UK) using a laboratory-made size ladder. The AFLP patterns of each parent strain were compared based on fragment sizes, and all polymorphic markers present in one parent and absent in the other were scored.

Development and analysis of SSR markers SSR markers were designed using the SSRPrimer program [START_REF] Jewell | SSRPrimer and SSR Taxonomy Tree: Biome SSR discovery[END_REF], which combines the algorithm Sputnik, to identify SSRs in the genome sequence, and Primer3, to design oligonucleotide primers to amplify the region corresponding to the SSR. For a particular sequence supercontig, the optimal SSR marker was selected based on the repeat type (di-and tri-nucleotide repeats were favoured), the number of repeats (over 20 repeats if possible) and the size of the PCR product (between 150 and 400 bp). Potential SSR markers were then tested by carrying out a simulated PCR against the complete genome sequence using the program e-PCR [START_REF] Schuler | Sequence Mapping by Electronic PCR[END_REF]. Primer pairs that were predicted to amplify more than one genomic region were eliminated and an alternative marker was selected for that supercontig.

PCR reactions for SSR genotyping were prepared on both Biomek NX (Beckman) and Star (Hamilton) liquid handling platforms. Adapting the method of [START_REF] Schuelke | An economic method for the fluorescent labeling of PCR fragments[END_REF], the 5 µl PCR reactions contained 12-15 ng of template DNA diluted in sterile Millipore water, 1 µl of 5x GoTaq reaction buffer, 0.25 µl of dimethyl sulphoxide, 10 nmol MgCl 2 , 0.5 nmol of each dNTP, 0.2 pmol of the forward primer (which included a 19 nucleotide tail corresponding to a sequence of the M13 bacteriophage), 2 pmol of the reverse primer, 1.8 pmol of the fluorescence-marked M13 primer and 0.2 units of recombinant GoTaq-polymerase (Promega, Charbonnieres, France). The PCR reactions were carried out on a 384 well plate PTC-115 thermocycler (Bio-Rad). The PCR protocol comprised an initial denaturation step at 94ºC for 4 min, followed by 12 touch-down cycles involving denaturation at 94ºC for 30 seconds; annealing at 65-54ºC for 1 min (decreasing by 1°C with each cycle) and extension at 72ºC for 30 seconds, then 25 cycles of denaturation at 94ºC for 30 seconds, annealing at 53°C for 1 min and extension at 72ºC for 30 seconds, with a final extension step at 72°C for 10 min. Two or three 384-well PCR products were simultaneous pooled and diluted 1:50 to 1:90 in 70% formamide supplemented with GenScan-500 LIZ Size Standard (Applied Biosystems, Foster City, USA). Electrophoresis and allele detection was carried out on an ABI3130xl Genetic Analyser (Applied Biosystems) with 36 cm capillaries. Output was analysed with Genemapper version 4.0 (Applied Biosystems). All marker data were verified manually by visual inspection to eliminate any errors introduced by the automatic allele assignation.

Linkage map construction

The fit of marker-allele segregations to the expected 1:1 segregation ratio was tested by a χ 2test (P = 0.05). Genotyping data were assembled in a unique file to elaborate the genetic linkage map. MAPMAKER/EXP 3.0 was used to build the genetic linkage map [START_REF] Lander | MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations[END_REF]. A minimum logarithm of odds (LOD) score of 4.0 with a maximum genetic distance of 50 cM was first used to associate SSR loci into initial linkage groups. Then, the LOD score was reduced to bridge some intervals. To determine the correct marker order within the linkage groups, multipoint analysis was performed using ''order'', "try" and "map" commands for groups containing more than 5 loci and using "comp" for groups containing less than or equal to 5 loci. Genetic distances were calculated according to the Kosambi formula.

Orientation of supercontigs on the genetic linkage map

When more than one marker had been mapped for a single sequence supercontig, it was possible to orientate the supercontig relative to the genetic map, provided that there had been recombination between the markers. The genes along each supercontig have been given locusID numbers that run sequentially (for the most part) along the supercontig. Supercontigs in which the genes with the lowest locusID numbers (i.e. at the "start" of the supercontig) were orientated towards the top of the linkage group (linkage group orientation as in Fig. 1) were defined as having a "+" orientation, whilst supercontigs with the opposite orientations were denoted by "-".

Results

Identification of compatible, polymorphic parental strains

The Ectocarpus genome sequence was generated using the male strain Ec 32, a meiotic offspring of a field sporophyte from San Juan de Marcona, Peru [START_REF] Peters | Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus[END_REF]. To identify a suitable female strain for the generation of the genetic map, 119 individuals were isolated from Peru and northern Chile and one of these strains, Ec 568, from Arica, Chile, was found to be fertile and compatible with Ec 32. The thalli raised from zygotes were capable of meiosis. Meiospores from one of these thalli (Ec 569) developed into female and male gametophytes confirming its heterozygosity. An amplified fragment length polymorphism (AFLP) analysis using 51 primer combinations was carried out on the two crossed strains to estimate the level of polymorphism between the two genomes. A total of 1,186 AFLP fragments were analysed. Of these, 703 fragments were amplified from both parents, indicating that these represented monomorphic regions of the genome, 170 were only amplified from Ec 32 and 313 only from Ec 368. This analysis indicated a high level of polymorphism (41%) between the two parental strains and these strains were therefore retained for the construction of the genetic map. There was a bias towards the detection of polymorphic fragments in the Ec 568 strain compared to Ec 32 (65% of the polymorphic fragments were found in the former).

Construction of a genetic map for Ectocarpus

One of the major aims of this project was to improve the large-scale assembly of the Ectocarpus genome by anchoring supercontigs onto linkage groups. To do this, the sequence of each supercontig was analysed using the SSRPrimer program [START_REF] Jewell | SSRPrimer and SSR Taxonomy Tree: Biome SSR discovery[END_REF] to identify a set of candidate SSRs for each supercontig. A two-stage marker selection process was then employed to maximise coverage of the longest supercontigs (i.e. to maximise the length of DNA sequence anchored per marker). In the first round 576 markers were selected from amongst the 624 longest supercontigs and tested for polymorphism. In the second round, an additional 576 markers were tested, including both alternative markers for supercontigs that did not produce usable markers in the first round and markers for additional supercontigs. The SSR markers to be tested were selected based on several criteria (see material and methods for details) and simulated PCRs were carried out against the complete genome sequence using the program e-PCR [START_REF] Schuler | Sequence Mapping by Electronic PCR[END_REF] to determine whether each oligonucleotide pair could be expected to amplify a single region of the genome. Testing for polymorphism by PCR amplification was only carried out using oligonucleotide pairs that fulfilled this criterion. A total of 1,152 markers were genotyped using DNA from the two parental lines. Of these, 522 (45%) were found to be polymorphic but 115 of the polymorphic markers had to be excluded because they did not give signals of sufficiently strong intensity or because the oligonucleotides amplified non-specific products or multiple loci. The remaining 407 oligonucleotide pairs were then used to genotype a population of 60 haploid F 2 progeny that had been generated by crossing the two parental lines.

To produce this segregating population, an F 2 population of haploid gametophytes was produced from the hybrid, diploid F 1 sporophyte (Ec 569) created by crossing Ec 32 and Ec 568 (see above). This was done by first isolating individual unilocular sporangia from Ec 569 and then isolating single gametophytes that germinated from the meiospores released by the sporangia. Each individual of the F 2 population was isolated from a different unilocular sporangium to ensure that each member of the population was derived from a distinct meiotic event. In Ectocarpus, unfertilised gametes can germinate parthenogenetically to produce haploid partheno-sporophytes. These partheno-sporophytes can then reproduce asexually via mito-spores. By exploiting this phenomenon, we were able to maintain each haploid line in partheno-sporophyte form. In order to obtain good quality, reproducible SSR genotyping data, we found that it was necessary to use CsCl purified DNA.

During the genotyping of the 60 F 2 progeny, one of the primer pairs amplified two polymorphic regions, providing a second unlinked marker (M_00X, Fig. 1) in addition to the marker for which it was designed. This additional marker was not predicted by the PCR simulation, indicating that it corresponds to a region of the genome that has not been sequenced. In all, therefore, 408 SSR markers were mapped against the two parental lines and the 60 F 2 progeny (Supplementary tables S2, S3). All the markers amplified fragments from both parents. The SSR markers allowed robust genotyping, on average each SSR marker was genotyped in 98.4% of the individuals of the population (i.e. the two parents plus the 60 F 2 progeny).

A minimum LOD score of 4.0 with a maximum genetic distance of 50 cM allowed us to obtain 35 linkage groups initially. We then used an LOD score of 3.5 to bridge some of the intervals. The final map consisted of 406 markers mapped to 34 linkage groups: 26 of which were between 47 and 170 cM long and 8 of which were between 2 and 23 cM long. Together, the 34 linkage groups span a total distance of 2,459.3 cM, with an average distance of 6.6 cM between adjacent markers (Fig. 1, Tables 1,2). If twice the average marker spacing is added for each linkage group (to account for the regions beyond the terminal markers at each end of the linkage group) this gives a total genome length of 2,908.8 cM. The 26 longest linkage groups span 2,350.7 cM with an average distance between markers of 6.7 cM. The 8 short linkage groups span 108.6 cM with an average distance between markers of 4.9 cM.

Three hundred and twenty five supercontigs were positioned on the genetic map, representing a total of 137 Mbp (70.1% of the genome sequence). Fifty-nine of the supercontigs were mapped using more than one marker (between 2 and 4 markers per supercontig, 139 markers in all). In all cases, physically linked markers mapped to adjacent positions on the genetic map. The genetic map was therefore strongly supported by the physical data for these markers. Forty of the 59 supercontigs could be orientated relative to the adjacent supercontigs using the linkage data (Table 3). An additional four supercontigs were orientated by combining linkage data with the presence of a gene that spanned a pair of supecontigs (see below). Comparison of the genetic and physical distances between these markers indicated that one cM was equivalent to 53.9 kbp. However, it must be noted that this relationship was highly variable depending on the pair of markers that was analysed, giving values ranging between 3.3 and 178.5 kbp/cM when individual pairs of markers that exhibited recombination were analysed.

Three markers were mapped for the longest supercontig (sctg_0000) but this region was not linked to any other supercontigs. This may be because this supercontig represents a discrete linkage group, but it is also possible that the markers used were not close enough to the extremities of the sequence (the external markers were 1,233 and 2,000 kbp from each end of the supercontig, respectively).

A small proportion of the loci showed segregation distortion (by a χ 2 -test, P≤0.05) in the segregating mapping population (20 loci on 8 linkage groups, representing 4.9% of the 406 mapped loci, Table 4). Most of these loci (60%) were widely distributed in the genome, being located on seven different linkage groups. However, eight of the loci with skewed segregation were clustered, covering nearly half of linkage group 33. This cluster comprises loci exclusively favouring the alleles of the male parent, Ec 32. Considering the small number of such loci, we chose to keep them in the data set, since they would not have had a significant skewing effect on the map.

Three markers exhibited evidence of a mutation occurring within the microsatellite sequence during the decent from the parents to the progeny (Table 5). For markers M_467 and M_030_4 the new mutant allele was different to both parental alleles and it was possible to determine which parental allele had mutated based on the segregation ratios. For marker M_032_3 however, the allele of the male parent appears to have mutated to have the same sequence as the allele of the female parent. This marker could not be exploited. Note that an alternative hypothesis is that M_032_3 corresponded to a locus exhibiting particularly strong segregation distortion.

The extremities of supercontigs that had been mapped to adjacent sites on the genetic map were analysed for the presence of genes that spanned the two supercontigs. Seven split genes were found including, for example, the Ectocarpus Rad50 gene (Table 6). Identification of these seven supercontig-spanning genes allowed the 14 supercontigs carrying the fragments of the genes to be orientated with respect to each other. Two of the supercontig pairs, on linkage groups 14 and 33, could also be orientated with respect to their linkage groups by combining this information with mapping data.

Sequencing of the genome of strain Ec 32 identified an inserted viral genome (Cock et al. submitted), closely related to the Ectocarpus virus EsV-1 [START_REF] Delaroque | The complete DNA sequence of the Ectocarpus-siliculosus-Virus Genome[END_REF]. Comparison of the inserted virus with EsV-1 indicated that a region of the genome surrounding the putative integrase gene EsV-1-213 had been deleted from the former. A homologue of EsV-1-213 was identified on a different supercontig suggesting either that this gene had integrated separately at a separate locus or that this region of the virus had transposed after integration into the host genome. Analysis of the genetic map indicated that these two fragments of the genome are in two different linkage groups, the majority of the viral genome in supercontig 0052 being located on linkage group 16 whilst the EsV-1-213 homologue on supercontig 0371 mapped to linkage group 23.

Discussion

In this study, we report the construction of a genetic map for the brown alga E. siliculosus, based on a cross between two highly polymorphic parental strains. Only a small number of genetic maps have been generated for species outside the opisthokont and plantae lineages. Within the stramenopile (or heterokont) lineage, to which the brown algae belong, molecularmarker-based maps have been reported for the distantly related oomycetes (van der [START_REF] Van Der Lee | AFLP linkage map of the oomycete Phytophthora infestans[END_REF][START_REF] May | Inheritance and mapping of 11 avirulence genes in Phytophthora sojae[END_REF][START_REF] Sicard | A genetic map of the lettuce downy mildew pathogen, Bremia lactucae, constructed from molecular markers and avirulence genes[END_REF][START_REF] Van Der Lee | High-density genetic linkage maps of Phytophthora infestans reveal trisomic progeny and chromosomal rearrangements[END_REF] and a tentative linkage map has recently been published for the kelp Laminaria japonica [START_REF] Yang | Construction and characterization of a tentative amplified fragment length polymorphism-simple sequence repeat linkage map of Laminaria (Laminariales, Phaeophyta)[END_REF].

The Ectocarpus genetic map consists of 26 large linkage groups and eight smaller linkage groups. The smaller linkage groups may coalesce with the larger linkage groups if additional markers were to be added to the map, and this process could be facilitated by exploiting the existing data. For example, all eight of the small linkage groups have large supercontigs (i.e. 400 kbp or larger) at at least one end, suggesting that densification of markers within these supercontigs could allow them to be linked to the larger linkage groups.

The number of linkage groups agrees reasonably well with the estimated number of 25 chromosomes reported for E. siliculosus by [START_REF] Müller | Untersuchungen zur Entwicklungsgeschichte der Braunalge Ectocarpus siliculosus aus Neapel[END_REF][START_REF] Müller | Generationswechsel, Kernphasenwechsel und Sexualität der Braunalge Ectocarpus siliculosus im Kulturversuch[END_REF]. The genetic map integrated 406 (99.5%) of the 408 markers that were genotyped, indicating that it covers a large majority of the genome. There are however regions where additional markers would significantly improve the map. The SSR markers used were robust, each providing genotype information for nearly all the individuals of the segregating population. The use of 140 markers that were physically linked to at least one other marker allowed a rigorous evaluation of the map. No conflicts were identified between the physical and genetic information for any of these markers.

About 5% of the mapped loci showed a distorted segregation pattern. Most of the distorted loci were scattered around the genome but eight loci clustered together on linkage group 33. A small percentage of markers are expected to show distorted segregation ratios as a result of random fluctuations but in the majority of cases the distorted segregation patterns observed probably reflected real biases in the relative transmissibility of the two alleles. Production of meiospores in the unilocular sporangia of the sporophyte involves a single meiotic division followed by a series of mitotic divisions that give rise to about 100 meiospores in each sporangium. These spores are then released and germinate to give rise to the gametophyte generation. Distorted segregation patterns may have caused by alleles that caused a reduced rate of cell division within the unilocular sporangium or negative effects on meiospore germination or growth of the gametophyte. Presumably, such effects would have been due to epistatic interactions between alleles at more than one locus because the presence of a haploid generation in the life cycle should effectively select against deleterious factors due to single loci. Moreover, incompatible combinations of alleles at different loci might have been expected in the mapping population, given the high level of polymorphism between the two parental strains (41% according to the AFLP analysis). Note that aberrant segregation patterns suggesting the occurrence of translocations and aneuploidy have been described in another stramenopile group, the oomycetes [START_REF] Judelson | Genetic and physical variability at the mating type locus of the oomycete, Phytophthora infestans[END_REF][START_REF] Carter | The detection of nonhybrid, trisomic, and triploid offspring in sexual progeny of a mating of Phytophthora infestans[END_REF][START_REF] Judelson | Multiple loci determining insensitivity to phenylamide fungicides in Phytophthora infestans[END_REF][START_REF] Van Der Lee | Chromosomal deletion in isolates of Phytophthora infestans correlates with virulence on R3, R10 and R11 potato lines[END_REF][START_REF] Dobrowolski | Non-Mendelian inheritance revealed in a genetic analysis of sexual progeny of Phytophthora cinnamomi with microsatellite markers[END_REF][START_REF] Van Der Lee | High-density genetic linkage maps of Phytophthora infestans reveal trisomic progeny and chromosomal rearrangements[END_REF].

Three of the markers used showed evidence of mutations occurring within the microsatellite region during the establishment of the mapping population. This represents a frequency of detectable mutations at each SSR of 0.37%.

The Ectocarpus genetic map, and the population of individuals that was used to generate it, represent important resources for future genetic analyses in this species. The map will allow new genetic mutations to be mapped to linkage groups, providing information about the positions of the corresponding genes in the genome and a starting point for positional cloning projects. In addition, the highly polymorphic mapping population could be exploited to screen directly for genetic factors that, for example, affect responses to environmental modifications. The haploid nature of the individuals permits direct screening for phenotypes associated with genetic polymorphisms without the confounding effects of dominance effects between alleles. Moreover, the sequence-tagged molecular markers allow phenotypic differences to be associated with specific regions of the sequenced genome. Another strong point of this system is that the population can be stably maintained in the haploid state so that phenotypic screens can be carried out at any time directly on an identical copy of the original mapping population.

The genetic map has also provided important information about the Ectocarpus genome, allowing 70.1% of the genome sequence (137 of the 214 Mbp) to be anchored onto linkage groups and, thereby, providing a large-scale view of the genome's structure. This information has been used to construct pseudochromosomes (by concatenating adjacent supercontigs) and to carry out broad analyses of genome composition including analyses of gene and transposable element densities and GC content (Cock et al., submitted). Analysis of the pseudochromosomes did not identify any clear, large-scale heterogeneity, although a small number of pseudochromosomes were either unusually rich in transposable elements (linkage groups 29 and 30) or had an unusually low density of genes (30 and 32;Cock et al.,submitted). *Indicates the 26 longest linkage groups. †Indicates the eight shortest linkage groups.

Tables

Table 3. List of supercontigs that have been orientated on the genetic map.

Linkage group

Supercontig Orientation

LG03

sctg_129 +* sctg_293 + LG04 sctg_6 - sctg_32 - LG05 sctg_268 - LG06 sctg_7 - sctg_214 - LG08 sctg_10 - sctg_23 - LG09 sctg_97 - sctg_27 + LG10 sctg_56 + LG11 sctg_25 + sctg_14 + LG12 sctg_16 - LG13 sctg_20 + sctg_199 + sctg_53 + LG14 sctg_171 - sctg_464 - sctg_303 + LG15 sctg_82 + sctg_24 - sctg_40 + LG16 sctg_62 - sctg_28 + sctg_86 + LG17 sctg_38 + sctg_29 - LG18 sctg_46 + sctg_327 - LG19 sctg_30 + LG20 sctg_122 + LG21 sctg_84 - LG22 sctg_98 - LG26 sctg_130 + LG28 sctg_244 + LG30 sctg_96 - sctg_105 - LG31 sctg_164 + LG33 sctg_121 + sctg_335 + sctg_425 - sctg_367 -
*Supercontig is orientated with the lowest LocusID numbers towards the top of the linkage group.

Figures

Fig. 1. Genetic linkage map of E. siliculosus. The map consists of 34 linkage groups. The positions of SSR markers are indicated to the right of each linkage group, with the prefix "M" for marker followed by the number of the supercontig that contains the SSR and finally, in some cases, a suffix to distinguish markers that originated from the same supercontig. Numbers to the left indicate the map distances, in cM, between the intervals given by the lines that cross the vertical bar. The cumulative distance between all the markers mapped to each linkage group is given in brackets. The 24 markers that exhibited segregation distortion are shown in bold. Note the cluster of eight of these markers on linkage group 33.

Table 1 .

 1 General statistics for the E. siliculosus genetic linkage map.

	All linkage	26 longest	8 shortest
	groups	linkage	linkage
		groups	groups

of markers genotyped 408 Number of markers located on the map 406 Number of markers showing segregation distortion 24 Average % of individuals genotyped by each marker 98.4 Number of supercontigs mapped 325 Amount of sequence anchored on the map (bp) 137249116 Mean number of markers per supercontig 1.25 Number of markers that are on a supercontig with another marker 139 Number of supercontigs with more than one marker 59 Number of supercontigs that could be orientated 40Table 2 .

 2 Statistics for individual linkage groups of the E. siliculosus genetic linkage map.

	20 †	22.0	6		5	4.4
	21*	60.3	11		8	6.0
	22*	53.8	7		5	9.0
	23*	54.6	13	11	4.6
	24*	59.7	10	10	6.6
	25*	76.1	10	10	8.5
	26*	135.6	22	18	6.5
	27*	123.8	12	11	11.2
	28 †	8.7	5		4	2.2
	29 †	22.9	5		5	5.7
	30*	95.4	20	17	5.0
	31 †	3.4	3		2	1.7
	32 †	1.8	3		2	0.9
	33*	95.3	14	12	7.3
	34 †	7.0	2		2	7.0
	All linkage groups
	total	2459.3	406	325
	mean	72.3	11.9	9.6
	min	1.8	2		1
	max	169.9	25	20
	*26 longest linkage groups
	total	2350.7	376	301
	mean	90.4	14.5	11.6
	min	47.0	7		5
	max	169.9	25	20
	†8 shortest linkage groups
	total	108.6	30	24
	mean	13.6	3.8	3.0
	min	1.8	2		1
	Linkage max	Length of linkage 22.9	Number of 6	Number of	5	Average
	group	group (cM)	markers	supercontigs	distance
			mapped	mapped		between
						two
						markers
	1 †	21.6	3		1	10.8
	2*	169.9	18	18	10.0
	3*	122.5	21	16	6.1
	4*	95.4	14	10	7.3
	5*	90.8	11	10	9.1
	6*	83	14	11	6.4
	7 †	21.2	3		3	10.6
	8*	75.4	11		9	7.5
	9*	114.3	19	15	6.4
	10*	47.7	11		8	4.8
	11*	108.5	18	13	6.4
	12*	67.6	9		7	8.5
	13*	47	11		7	4.7
	14*	102.5	14	11	7.9
	15*	124.4	17	12	7.8
	16*	128	25	20	5.3
	17*	97.6	18	14	5.7
	18*	63.4	19	12	3.5
	19*	58.1	7		6	9.7

Table 4 .

 4 Markers that showed distorted segregation.

	Marker Linkage	Ec 32	Ec 568	Parental allele
		group	(male)	(female)	over-
					represented
	M_000_8	1	19	35	Ec 568
	M_008	2	22	38	Ec 568
	M_131	11	19	41	Ec 568
	M_155	11	21	38	Ec 568
	M_086_1	16	15	28	Ec 568
	M_136	16	20	37	Ec 568
	M_065_3	21	38	22	Ec 32
	M_179	21	38	22	Ec 32
	M_368_2	21	38	21	Ec 32
	M_019	25	39	20	Ec 32
	M_178_3	25	38	22	Ec 32
	M_103_2	27	38	22	Ec 32
	M_035	33	38	22	Ec 32
	M_054	33	40	17	Ec 32
	M_121_2	33	43	12	Ec 32
	M_121_3	33	42	18	Ec 32
	M_240	33	41	18	Ec 32
	M_328_1	33	41	19	Ec 32
	M_343	33	43	16	Ec 32
	M_425	33	41	19	Ec 32

Table 5 .

 5 Markers that mutated during the establishment of the mapping population.

	Marker Type	32M allele	568F allele	mutant allele
	M_467	tri	0 (16 repeats) 26 (15 repeats) 34 (14 repeats)
			31 (32
	M_030_4	di	repeats)	8 (16 repeats)	20 (14 repeats)
	M_032_3	di	1 (35 repeats) 59 (36 repeats) (= 568F allele)

Table 6 .

 6 List of genes that span two supercontigs, which were mapped next to each other on the genetic linkage map.

	Linkage

group Upper supercontig (and gene locusID) Lower supercontig (and gene locusID) Map distance between the markers on the two supercontigs Predicted gene function or protein domains present

  Supplementary tableS1. Numbers of polymorphic and monomorphic bands detected with different primer combinations in the AFLP analysis of the two parental strains.

	EcoRI primers	MseI primers	monomorphic polymorphic Es 32 polymorphic Es 568
	EcoRI + AAA	MseI + CAT		10	3	9
			MseI + CAG		23	10	11
			MseI + CAC		12	3	5
			MseI + CTA		23	6	26
			MseI + CTT		6	0	2
			MseI + CTG		6	0	4
			MseI + CGC		11	2	9
	EcoRI + AAT	MseI + CAT		12	7	3
			MseI + CAG		19	1	4
			MseI + CAC		17	2	7
			MseI + CTA		6	1	1
			MseI + CTT		11	1	7
			MseI + CTG		15	0	6
			MseI + CGC		20	2	13
	EcoRI + ATA	MseI + CAT		5	2	2
			MseI + CAC		13	7	10
			MseI + CTA		10	0	6
			MseI + CTT		18	6	3
			MseI + CTG		17	1	5
			MseI + CGC		14	1	4
	EcoRI + ATT	MseI + CAT		9	1	5
			MseI + CAG		6	1	0
			MseI + CAC		15	1	0
			MseI + CTA		21	2	16
			MseI + CTT		16	9	11
			MseI + CTG		8	2	8
			MseI + CGC		9	2	1
	EcoRI + ACC	MseI + CAT		8	1	0
			MseI + CAC		8	2	8
			MseI + CTA		2	0	2
			MseI + CTT		40	7	18
			MseI + CTG		32	6	6
	EcoRI + ACA	MseI + CAG		3	2	7
			MseI + CAC		14	1	5
	LG02 LG05	sctg_203 (Esi0203_0057) MseI + CTA MseI + CTT sctg_211 (Esi0211_0048) MseI + CTG MseI + CGC	15 (Esi0465_0002) sctg_465 11 sctg_338 (Esi0338_0032) 7 7	8 0 2 0 1 1	3 3 4 Glycosyl transferase, Rad50 family 48 3
	sctg_198 EcoRI + ACG MseI + CAT	sctg_403	7	0	Acetyl-CoA 2
	LG07	(Esi0198_0003) MseI + CAG	(Esi0403_0027) 12	0 6	carboxylase 5
			MseI + CAC		14	1	Di-copper centre-2
	LG08 LG13 LG30 EcoRI + AGT sctg_406 (Esi0406_0025) MseI + CTA sctg_303 (Esi0303_0037) MseI + CTT MseI + CTG sctg_560 (Esi0560_0001) MseI + CGC MseI + CAT sctg_335 MseI + CAC	sctg_203 (Esi0238_0001) 27 sctg_464 (Esi0464_0017) 1 14 7 sctg_357 (Esi0357_0001) 14 sctg_425 16	0 2 7.2 1 1 0 0 3 3	containing domains 9 (IPR008922) 2 tRNA pseudouridine 4 synthase 6 5 Ubiquitin-protein ligase Radical SAM domain 9
	LG33	(Esi0335_0025) MseI + CTA	(Esi0425_0023) 58	9.1 18	(IPR002792) 12
			MseI + CTT		17	3	6

Acknowledgements

This project was supported by grants to SH from the Conseil Générale de Finistère and to GYC from the Korean Research Foundation and by support from the GIS Europôle Mer, the Centre National de Recherche Scientifique, the European Union network of excellence Marine Genomics Europe, the Europole Mer and the University Pierre and Marie Curie. We would also like to acknowledge the help of Delphine Scornet, Sylvie Rousvoal, Laurence Dartevelle, Sarah Stewart, Morgan Perennou, Sarah Romac, Serinde J. van Wijk, Marie Laure Guillemin (all at the Station Biologique de Roscoff, France; SJvW is now at Bangor University, UK, MLG is now at the Pontificia Universidad Católica de Chile, Santiago, Chile) and Gwenn Verplancke (INRA, Le Rheu, France).

 S3. Results of the genotyping for the segregating population.

(see separate file)


J.-M Thiriet
email: jean-marc.thiriet@ujf-grenoble.fr

D Lubineau
email: denis.lubineau@ujf-grenoble.fr

P Benech

E Novakov
email: emil.novakov@ujf-grenoble.fr

A Derioz
email: anna.derioz@ujf-grenoble.fr

J.-M Terriez

Implementation of an International "Professional" Bachelor's Degree in Wireless Networks and Security in Grenoble

The 'Professional' Bachelor's Degree programmes "Networks and Telecommunications" specialising in "Wireless Networks and Security" has existed since 2005 in Grenoble. This programme is actually the 5 th and 6 th semesters of a three-year degree. In order to encourage international exchanges within this programme, in September 2007 we launched as part of it an "international" group of students (fourteen) with courses taught in English, in order to attract to France both/either non Frenchspeaking European students within the framework of the ERASMUS exchanges, and/or non-European students. This is an innovative pilot project in France since it is the first time a complete academic year of tuition has been available in English at Bachelor's degree level in the technological fields of study.

I. INTRODUCTION

The Professional Bachelor's Degree "Networks and Telecommunications" specialising in "wireless networks and security" started in Grenoble in September 2005 (Frenchspeaking programme). In the French system, this programme concerns the last year at bachelor' degree level (3 years and 180 ECTS) which means the 5 th and 6 th semesters of the bachelor's degree programme.

The first group of students for this last year of the Bachelor's Degree programme (French-speaking class only) who left in June 2006 was composed of 32 students, and the second one in June 2007, of 25 students, which corresponded to our plans. In September 2007, an apprenticeship group started (part-time in a company and part-time in the university), so the figures for the French-speaking courses are 24 students in the initial traditional course and 19 in the apprenticeship one in 2007-2008.

The aim of the French Professional Bachelor's Degree is to give students a good level in scientific and technological skills. However another important part of the programme highlighted by French companies, is to prepare students to manage small projects with all the components: planning, customer/supplier relations, etc.

In order to develop international exchanges, especially with European countries where French is not so widespread in the technological fields, we decided to set up an international group, reduced in numbers, 14 students, with the same modules taught in English. This course began in September 2007, and concerns only, as mentioned before, the 5 th and 6 th semesters.

The purpose of this paper is to describe our experience concerning this international course. In the first part we describe how it is organised, the points which should be taken into account, some difficulties which have been encountered and some solutions or ideas, for colleagues who plan to organise such a degree programme.

The second part of the paper describes the targets of the project. In the third part how the course has been adapted to the international environment is discussed. The fourth part presents in particular the induction week. The fifth part describes the recruitment of students, based on a network of partners at European level. The sixth part presents the teacher mobility aspect of the project, since the Staff-Erasmus exchange programme is used in order to emphasise the international context of the programme. With Professional Bachelor Degrees, one of the main purposes is to give the students the opportunity to have a professional experience in a company or in a research laboratory. Therefore the seventh part presents this aspect. The eighth part is dedicated to the external communication strategy adopted -how to inform about or advertise the existence of our course. The ninth part points out some difficulties concerning particularly the problems of degree equivalence and recognition. The tenth and final part deals with propositions for the future… II. TARGETS OF THE PROJECT The idea of this international course came from the fact that today it is becoming increasingly difficult to attract foreign students onto our scientific and technical degree programmes and in particular Europeans, due to the difficulty of finding students in our disciplines who have good communication skills in French. Nevertheless, many European students are interested in France as such and would like to come to France to study.

It is important at this point to take into consideration the fact that traditionally, we succeed in attracting foreign students from French-speaking countries (Francophonie) mainly from Africa and the Middle-East, and also some students from Latin America within the framework of exchange programmes between France and certain Latin American countries (2 students from Venezuela were in Grenoble in 2007-2008, for instance). This strategy which consists in being 'open' towards "la Francophonie" is very important for us and is adopted within our traditional Frenchspeaking class.

To attract more European students the only option available to us is to lecture in English, since such students are not able to attend a course given in French, despite their willingness to come to France. It was thus decided to open an international class, composed ideally of more than one third ERASMUS-European students, one third students from other non Frenchspeaking countries (Asia, America, English-speaking Africa), and one "small" third made up of French students who are keen to work in this international environment. It is important for us to have some French nationals in this group to provide the link between the international class and France, since the course is in France.

Even though the courses are actually taught in English, an introductory course on French language (FLE: Français Langue Etrangère, French as a Foreign Language) together with the French cultural and economic environment is offered to the foreign students; the idea is that these students, once back home in their respective countries, will want to look further into their partnership with France. Proposing our course to French students as well as foreigners enables us to give it a truly international dimension. This is essential if we wish to initiate the non-French speakers to French culture and the French students to European mobility, and also intercultural partnerships… The French students in the international group have the opportunity to carry out their industrial placement (in a company or in a research laboratory) during the second semester in a foreign country.

III. ORGANISATION OF THE PROGRAMME

In order to internationalise our course, the first step was to define the contents, which sometimes required us to adapt the existing French course.

The strategy adopted is as follows: -Concerning the "General courses", we adapted in the following way:

 The English course C1 was initially replaced by a course on FLE for the non-French-speaking students and by an introductory course to the language of the country where the student will carry out his/her industrial placement for the French-speaking students.

Finally we decided later on to keep the English course, because the students needed to have some support in English, even if their level is quite good, in order to improve their skills. Concerning the "Fundamental scientific and technological courses" and "Specialised scientific and technological courses", the tutorials, lectures and practicals are taught in English. There is no specific aspect except for the fact that we use the English version of operating systems, both for server and client machines. Nevertheless we keep the use of "azerty" keyboards which has proved to be of no great difficulty for the students… Table . 3 Specialised scientific and technological courses, with the number of hours per course and the corresponding ECTS, for the 5 th semester Concerning these courses, sections of the tutorials are taught by European lecturers within the framework of ERASMUS Staff exchanges, i.e. 5-hours of tuition over a period of 3 to 4 days in Grenoble. The courses in question relate to networks, wireless networks, security, telecommunications and mobile telephony.

The 6 th semester is composed of three weeks of courses (roughly 60 hours for 4 ECTS) -courses C11 to C13, one course on database management systems, one introductory course on patent law and one languages course (English or, if it is possible, the language of the host country for the training period). The major part of the 6 th semester is dedicated to an industrial placement, as an opportunity for the student to apply , in real practical situations, the skills and knowledge acquired during the course. This placement can be carried out in a company, or in a research laboratory; the normal duration is 16 weeks for a total of 26 ECTS credits. The industrial placement is described below, in particular concerning international exchanges.

Two possibilities (options) are open to the students:  The first option consists in attending the courses in Grenoble only for the fifth semester of then going back to the home university in order to follow the sixth semester and pursue the syllabus there. In this case, they will follow the course in Grenoble for a semester within the framework of Erasmus exchanges but without being awarded the Professional Bachelor's Degree; they can however validate this semester as an "elective" as part of their course in their home institution.  The second option consists in attending the complete year, which allows the student to obtain the French Professional Bachelor's Degree if he/she has the necessary grades.

IV. WELCOME TO THE STUDENT: AN INDUCTION WEEK For this first experience of welcoming international students to Grenoble, we began the academic year with an induction week. The purpose was manifold:

 to assess the actual English level of the students, and the way they communicate,  to create relationships among the students (group cohesion),  to favour a good working partnership between students and lecturers,  to favour relationships between French and foreign students,  to organise a mentorship between French-speaking and non French-speaking students: the former helping the latter to resolve administrative problems (opening a bank account, finding accommodation, to getting possible additional grants for lodgings (Caisse d'Allocations Familiales)…). This induction week was organised as follows:  half a day for the "official welcome",  half a day dedicated to administrative aspects,  half a day dedicated to a social event: a walk up La Bastille, small hill with a beautiful panorama near Grenoble city centre,  the remaining 3 days were dedicated for each student to the preparation of a presentation about themselves, their motivation, their country, their country's academic system (fig. 1),…

Fig1Example from a student presenting his academic system V. RECRUITMENT OF STUDENTS In 2007-2008, 5 French students and 9 foreign students were signed up for the programme.The French students and some foreigners (generally the French-speaking ones) came through the filter of the "normal" registration procedure and were required to fulfill one of the following criteria regarding their level in English:

 TOEFL 520 (paper-based exam) or 210 (computerbased exam),  TOEIC 700,  IELTS 5.5,  BULATS 70.

They were also required to provide a letter of motivation explaining their reasons for wishing to attend the international course.

The European students were recruited through either the network of the "historical" partners of the Université Joseph Fourier, or the network of partners within the EAEEIE (European Association for Education in Electrical and Information Engineering, www.eaeeie.org) Association and the EIE-Surveyor (www.eie-surveyor.org) ERASMUS thematic network.

Of the 9 foreign students, among whom 5 originated from ERASMUS exchanges, in 2007-2008, there were:

 3 from the Technical University of Košice (Slovakia),  1 from Stadia University in Helsinki (Finland),  1 from the University of Technology of Silesia (Poland).

The other foreign students included one Greek, one Canadian, one Chinese, one Mongolian registered through normal channels. All these students were also required to fit the level of English described above. Concerning the European partnerships, in addition to the three partners who sent us students within the framework of the Erasmus exchanges, twenty-eight other partners expressed an interest in our proposal. Hopefully they will be real partners in the future.

As far as their scientific and technological level is concerned, each student profile was analyzed, in order to see if he/she can follow the courses in good condition. We had in 2007-2008 some difficulties with one student (who had a good theoretical approach but no practical experience.

VI. MOBILITY OF LECTURERS

One of the interests of this programme is to provide the students with an international or at least a European dimension. One of the ways to do so is to invite colleagues, from European partner institutions within the Erasmus Staffexchange programme, to come to Grenoble and teach part of the course to the students. The purpose of this Teacher mobility is twofold:

 to make the students aware of different viewpoints from a variety of European countries,  to further exchanges between lecturers, our case between the European colleagues and the Grenoble staff. ). The Finnish colleague who came is a specialist in communication skills in an international environment, and her conference was very much appreciated since the students on the international programme are working in an international environment, and also because we want them to be taught about these aspects and to gain the corresponding skills. The last aspect, outside of the ERASMUS Staff-exchange, is a partnership we have with EURES, which is a European network of employment agencies. The aim of this network is to emphasise the exchange of workers at European level, and particularly from the border countries, to give the possibility to people to cross the border in order to work in the neighbouring country. A French and a Czech colleague came to Grenoble for this purpose.

VII.

PLACEMENTS IN COMPANIES OR RESEARCH LABORATORIES For students interested (second option described at the end of section 3), the 6 th semester of this programme provides the opportunity for the student to spend 16 weeks in a company, or possibly in a research laboratory.

Concerning the international programme, we encourage students to be mobile, in particular the French to go abroad and the foreigners either to stay in France or to go to another country, different from their own. Thanks to the network of partners in our University and the one of the EIE-Surveyor network, more than 20 placements were available for the 14 students for 2007-2008.

The students were in the following countries:  6 in France (in 6 companies),  3 in the Czech Republic (in a laboratory, CTU),  1 in Germany (in the Fraunhofer Institute),  1 in Greece (in a company),  1 in Denmark (in a company),  1 in Canada (in a company),  1 chose option 1 (and so is back in his home university, in Finland, for the 6 th semester).

VIII.

COMMUNICATION STRATEGY In order to advertise this new syllabus, we contacted various European partners. The partners were contacted by email but also directly during international open days or conferences. That made it possible to identify specific aspects to be taken into account to make the project a success. We would emphasize at this level the fact that the direct contact with and direct involvement of people, as a result of former partnerships, at the teaching or the research levels, are really important and helpful, at least in the "launching" phase…

The strategy also concerns the advertising of this new programme through specialized organizations at the French level, such as SFERE and EduFrance.

SFERE is an organization linked to the French Ministry (Department) for Foreign Affairs dedicated to setting up, managing and monitoring of bilateral agreements between France and foreign countries (generally outside Europe) in order to give the opportunity to foreign students to attend courses in France.

EduFrance advertises through its website and also an annual paper directory the various programmes available in France for international students. There is specific information about English-taught programmes available in France. Our programme is thus now in the catalogue of the English-taught programmes offered in France.

IX. DIFFICULTIES ENCOUNTERED

Certain difficulties however appeared. Some were simply related to logistics (late launching of the project, motivation of the students when it came to taking the decision) but others were more fundamental.

The more structural difficulties are various:

 the students at Bachelor's degree level are perhaps less inclined to go abroad than the Masters students, they are indeed younger and less mature: we think that we should incite them to be motivated, and help in relaying the information about our course by colleagues in the partner universities is crucial,  problems with curricula compatibility (reintegration of the students into their original course at home after one semester spent in Grenoble) were raised by certain partners; this problem is a recurring problem raised by most teaching staff when there is a question of sending students abroad, or giving an equivalence when they return back home. It is important at this stage to consider that a student going abroad is benefitting from a highly interesting experience, as a student, in his field of competence, but also as a citizen; it is thus necessary to analyse the program as a whole and consider all the skills gained by the student abroad vs. the skills and knowledge required to follow the course, when the student is back in his/her home country; the preceding approach has more chance of success than an approach which consists in finding in the partner university an exact point-to-point equivalent course which is generally impossible to achieve. This point is discussed later on in the next part.  the problem of compatibility of courses also stems from the fact that a Bachelor's degree, in compliance with the Bologna model, may take, depending on the country or the institution, three years, three and a half years or four years; this point must of course be taken into account when setting up exchanges,  problems with diploma accreditation which prevent certain universities in certain countries from recognising semesters spent abroad,  difficulty also in finding work placements in companies in France for non French-speaking students. Several companies in France work in an international environment, in particular in the Grenoble area;

nevertheless, when it comes to recruiting a non Frenchspeaking student for a placement, most companies are generally reluctant to do so, arguing that it would be difficult for the student to interact with many people in the company in everyday life: this is due to the fact that English is not yet sufficiently widespread in France… X. SYNTHESIS AND DISCUSSION Several problems have been highlighted in the previous part. The main problem is equivalence (credits) and also accreditation [START_REF] Thiriet | Implementation for a US-European Trans-national Degree in Real-Time Software Engineering[END_REF]. These problems are discussed in the following paragraphs with some concrete propositions.

X.1 Equivalence and credits Concerning equivalence and credits, despite the interesting tool which ECTS (European Credit Transfer System) is [START_REF] Thiriet | Implementation for a US-European Trans-national Degree in Real-Time Software Engineering[END_REF] [START_REF] Adam | Principle of a Pan-European Credit Accumulation Framework: good practice guidelines[END_REF] [3], it is for the moment not easy to work with semesterial equivalence for mobility. First of all, we should remember that the ECTS is a tool which measures the actual workload of students, and the "grading" system which is used by ECTS is the following: A best 10%, B next 25%, C next 30%, D next 25%, and E next 10%. A distinction is made between the grades FX and F that are used for unsuccessful students. FX means: "fail-some more work required to pass" and F means: "fail -considerable further work required".

This method of giving levels (from A to E, and F et Fx) is statistically-based and relative. Its purpose is to be able to give the position of a student (who is in another institution) within a certain group -at least statistically representative -of students. It is not always the way ECTS credits are used … Consequently it is necessary to keep an actual grading system (such as the systems existing generally in HEIs, or a new standards to be invented [4]) and then to give ECTS correspondences. Another important aspect is that ECTS aims only at measuring the workload of the student, neither the time spent by the student with a teacher nor the level of qualifications obtained by the student is taken into consideration. These aspects should be emphasized in the future, for instance for the setting up of a Life Long Learning Accumulation system.

For our Professional Bachelor's degree, we used the workload in order to attribute ECTSs to the courses, these ECTSs are effectively used for ERASMUS exchange (option 1). The normal French grading system is used in order to deliver the French diploma, in the same conditions as for the students in the French-speaking programme.

X.2 Accreditation

The problem of accreditation should also be taken into account. In some countries and institutions and for some programmes, it is required to describe the complete syllabus, in detail, in order for the syllabus to be accredited. Under this assumption, it is really difficult to find the same courses in another institution and so to set up worthwhile international exchanges. A more pragmatic way is to base the accreditation on competence, this approach is strongly encouraged by the European Commission, within the framework of the Tuning Project [START_REF] Tuning | Tuning Educational Structure in Europe[END_REF].

X.3 A network of partners

We have understood from previous discussions that a network of partners is very important in such projects, with strong bilateral relationships as relays of information in partner institutions, based administratively on Erasmus bilateral agreements.

In order to facilitate exchanges and also to ease the accreditation procedures, it could be interesting to develop a multilateral exchange model. The exchanges could indeed be facilitated by the fact that they would not be based on a bilateral agreement but more on a multilateral base, which would give more choice to the students. The accreditation procedures could be eased within this framework, if we could organize within this multilateral basis some "compatible" areas of the syllabus. By compatible, we mean the organization and scheduling of these areas of syllabus more than the content itself. The contents could actually be different, it is also one of the interesting aspects of mobility [6] [7].

An idea for the implementation of the above concept consists in taking as a starting point the model of major and minor courses. Minor is an "under-specialty" or second specialty or elective which is proposed in certain programmes. We could imagine offering our semester (at present the 5 th semester of a Bachelor's degree programme) as an international semester within the syllabi of our European partners in similar fields of study. This approach is of interest for the following reasons:

 the semester could fit into any programme of study,  the accreditation procedures could take this aspect into consideration,  the problems of pre-requisites necessary for the students to follow courses when they go back to their home institution would be solved, since the semester proposed would be a minor. If the same "architecture" is implemented for minors by several partner institutions (network of partners) at the 5 th semester Bachelor level, these "international" minors could be proposed to students within the network, each student could then choose.

Proposing international semesters could thus solve certain remaining problems related to exchanges and thus encourage mobility. We could also imagine, with certain partners, standardizing and generalizing within a network the principle of the international semester minor, the students being able to choose from among the offers proposed….

Some points remain to be discussed, in order to generalize the approach: preparation of the students before mobility, the use of distant-based teaching resources, the possibility for students to follow some courses at a distance, once abroad, in order to have some complementary skills necessary to actually validate the semester. Nevertheless, this should be limited to perhaps a maximum of 4 ECTS credits in order to be realistic.

XI. CONCLUSIONS

This first year (2007-2008) was a good experience for our teaching staff on the programme. A good group of 14 students was recruited, among whom 9 foreigners, 5 of which did not speak French. Among the French students, 3 of them had been abroad in 2006-2007 on Erasmus exchange programmes and were particularly interested and motivated by the Englishtaught format of the proposed programme.

Two criteria were highlighted as requirements for the experience to be a success:

-the setting up of an international network of partners, in particular for student recruitment and industrial placements, -the creation of a strong teaching team, with proactive colleagues who are keen to be involved in the experience. A minimum number of colleagues is required -ideally one colleague per course is necessary. For our experience in Grenoble everything was covered except Project Management and Databases, here we found colleagues from another department of the university, these colleagues are now active members of our team… One difficulty which remains to be resolved, but on which we are at present working, is the fact that our final diploma is in French, as all official diplomas of the French Republic are. We are working on an accompanying document, with the translation of the Diploma and the Diploma Supplement.

Fig. 2

 2 Fig. 2 The group of students in 2007-2008

Table . 2

 . The legislation course C3 is taught in English, with a European orientation. To this end, a partnership has been established with the "Legal framework for the information society" European thematic network. An Italian and a Greek colleague came to Grenoble this year, within the ERASMUS teaching mobility exchange programme, to lecture on internet legislation with a European dimension. Fundamental scientific and technological courses, with the number of hours per course and the corresponding ECTS, for the 5 th semester

			Table. 1 General courses, with the number of hours per course and
			corresponding ECTS, for the 5 th semester
			This course also includes a course on the French and
			international economic	environment, consisting of
			conferences organized by the ANPE (Agence Nationale pour
			l'Emploi, the National French Employment Agency) within
			the framework of a European project (EURES, European
			Network of Employment Agencies), with talks by participants
			from a variety of European member countries.
			 The course C2 on French communication skills is
			replaced by a course on communication skills in
			English.
			Courses 5/6 : Fundamental scientific and	hours	ECTS
			technological courses
			C5 Interconnection of networks and their	60	4
			security
			C6 Telecommunications engineering	40	2
	Courses 1-4 : General courses	hours	ECTS
	C1: French language and civilisation and the	30	2
	French industrial environment
	C2: Communication skills	20	2
	C3: Introduction to Internet Legislation	20	2
	C4: Project Management	20	2

  The project management course C4 is also taught in English and is, as in the French course, closely linked to the academic project.

Table . 4

 . Programme of the 6 th semester

	Courses 11-14	hours	ECTS
	C11 Database management systems	30	2
	C12 Introduction to patent law	10	1
	C13 Language	20	1
	C14 Industrial placement in company or
	research laboratory

ACKNOWLEDGMENTS

The authors would like to thank all the partners, the EIE-Surveyor thematic network, our University and the IUT1 for their contribution, for their help with student recruitment, Erasmus staff exchanges and propositions of industrial placements.


Malika Ibrahim-Ouali

Frédéric Dumur

Archive for Organic Chemistry

Keywords: chalcones, steroids, enones, pregnenolone. Arkivoc 2019, part _, 0-

It was observed that the presence of heterocycles to steroids often leads to a change in their physiological activity and the appearance of new interesting biological precursors. Recent developments in the syntheses of steroidal chalcones and their derivatives are described herein. The biological activities of those steroidal derivatives for which data are available are given. The chalcone moiety shows great promise to allow us to develop novel molecules with characteristic functions.

Introduction

Steroids represent a large and important class of biologically active polycyclic molecules that are widely used in medicinal chemistry. [START_REF] Zeelen | Medicinal chemistry of steroids[END_REF][START_REF] Trager | [END_REF][START_REF] Biellmann | [END_REF] Even today, the total synthesis of steroids by new and more elaborate strategies continues to interest chemists. Thus, many total syntheses of biologically active steroids have been developed in recent decades. Interest in the total synthesis of such molecules continues to this day. 4,5 Chalcones (1,3-diphenyl-2-propen-1-one) are well known for their interesting biological properties. It is thus known that chalcones exhibit biological activities, such as anticancer, antioxidant, anti-inflammatory, 6,7 and antimicrobial activities. 8,9 Two phenyl rings and a , -unsaturated carbonyl system that connects them constitutes the structural base of chalcone. It has been shown that the nature of the substituents on the phenyl rings and their conformation in the central structure is at the origin of their biological activities. Some work has shown that reactions causing conformational changes, such as substitution on the double bond or epoxidation, can lead to a decrease in bioactivity. 10 The synthesis of hybrid compounds from the coupling of two or more natural products is at the origin of an inexhaustible new class of molecules with many structures. The objective of such syntheses is to combine the pharmacological properties of two or more biologically active molecules to increase or modulate these activities to obtain derivatives exhibiting new properties. 11,12 Previous works of literature have been reported that such structures as steroidal chalcones have been shown to bear a lot of different biological activities such as antimicrobial, anti-inflammatory, anticancer, antioxidant, and hypotensive activities. [13][14][15][16] This review presents the various syntheses of steroidal chalcones and their derivatives along with interesting biological activities, which have appeared in the literature in recent years. To the best of our knowledge and much to our surprise, there are no reports on this subject.

Synthesis of steroidal chalcones and derivatives

In 2007, Negi et al. 17 reported the synthesis of steroidal chalcones from estradiol. Some of these compounds have significant anticancer activity against certain human cancer cell lines. Estrone 1 was chosen as the starting aldehyde substrate to carry out the synthesis (Scheme 1). Thus, to prepare the aldehyde derivative 5, a protection reaction by methylation of the phenolic hydroxyl of 1 was first carried out using dimethyl sulfate and anhydrous potassium carbonate in anhydrous acetone under reflux. Protected estrone 2 was isolated with a yield of 91%. After a reaction of reduction using sodium borohydride in a mixture of methanol-chloroform (2: 1), the ketone of derivative 2 led to the corresponding alcohol 3 in a yield of 94% and an acetylation reaction using acetic anhydride in dry pyridine of this latter, provided the steroidal derivative 17-acetate 4 in a very good yield. A Vilsmeier formylation reaction, 18 using dimethylformamide and phosphorus oxychloride at 80 ° C resulted in the aldehydic substrate 5. The desired steroidal chalcones deprotected at the C-17 position were obtained by reacting the substrate 5 with various acetophenone derivatives in a solution of hydroalcoholic potassium hydroxide at 7%. Chalcones 6 and 14 were then acetylated with acetic anhydride to give the corresponding acetylated derivatives 7 and 15.

Scheme 1. Synthesis of steroidal chalcones 6-17.

One of the chalcone derivatives was further modified to the corresponding 1-indanone derivative 18 using the trifluoroacetic acid-catalyzed Nazarov cyclization reaction (Scheme 2). Indanone 18 was prepared in a yield of 23% by treatment in a sealed tube of chalcone 7 with trifluoroacetic acid for 4 h. 19 Some of these chalcones show significant anticancer activity against certain human cancer cell lines. This is the case with derivatives 9 and 18 which exhibit real activity against MCF-7, a hormone-dependent breast cancer cell line. Indanone 18 showed better activity than the parent chalcone against the MCF-7 breast cancer cell line. Using the erythrocyte as a model system, the most active chalcones were also tested for osmotic hemolysis. It has thus been shown that the fragility of erythrocytes is not modified by these biologically active chalcones. We can therefore consider these chalcones as non-toxic for normal cells. Scheme 2. Synthesis of the steroidal indanone 18 from chalcone 7.

In 2013, Sarma et al. 20 reported the synthesis of a new class of chalconoyl pregnenolones based on a Claisen-Schmidt reaction without solvent and under microwave activation conditions.

Cortisone, estrogen, testosterone, and progesterone among others are natural hormones biosynthesized from pregnenolone, a neurosteroid derived from cholesterol by biosynthesis in the adrenal gland and the central nervous system. 21,22 The condensation between the acetate derivative of pregnenolone 20 and the benzaldehyde 21 carried out without solvent and by microwave activation using 20 mol% of the catalyst, I2-Al2O3 led to the desired compound with a good yield of 86% after only 6 min of reaction at 250 W of microwave power. The authors were thus able to prepare sixteen new steroidal chalcones by condensation of compound 20 with different substituted benzaldehydes (Scheme 3). Furthermore, no by-product was observed, these clean reactions led to the desired chalcones with good yields of between 79 and 86%. Longer reaction times or an increase in the percentage of catalysts resulted in the decomposition of the desired product. In addition, the nature of the substituents on the aromatic ring of benzaldehyde does not affect the yield of the desired product.

Two fungal strains Aspergillus niger and Candida albicans and two bacterial strains Bacillus subtilis and Escherichia coli were used to test the synthesized derivatives 22-37. Some have interesting inhibitory activity. Furthermore, it has been observed that this activity is considerably reduced if the double bond is epoxidized. The presence of the unsaturated chalcone moiety, therefore, plays a significant role in this inhibitory activity.

In 2015, El Sayed Aly et al. 23 described the synthesis of steroidal chalcones from cholesterol in good yield. The starting chalcones were synthesized as described in Scheme 4. Intermediates 40a-b and 41a-b were prepared in average yields at room temperature by propargylation of chalcones 39 using propargyl bromide and the potassium carbonate as the base. The chalcone 40c was prepared by the Claisen-Schmidt condensation reaction of intermediate 42, previously propargylated as described above, in the presence of pdimethylaminobenzaldehyde. In doing so, the extinction of propargyl bromide as a quaternary ammonium salt by the dimethylamino group is prevented which would not have been the case if the chalcone engaged had been propargylated as described previously for compounds 40a-b and 41a-b.

Scheme 4. Synthesis of chalcones derivatives 40a-c and 41a-b.

Cholest-5-en-3β-ol 43 was activated as bromide in very good yield under Appel conditions, 25 which means treatment with CBr4/PPh3 to afford 3α-bromocholest-5-ene 44 due to inversion of the configuration at the C-3 carbon (Scheme 5). An SN2 substitution of the bromine atom of compound 44 with the N3 group was ensured by refluxing with NaN3 in dry DMF to afford 3β-azidocholest-5-ene 45 after inversion of the configuration again at the C-3 carbon. The product was isolated in good yield.

The target cholesterol-chalcone conjugates 46a-c and 47a-b were prepared by reacting 3β-azidocholest-5ene 45 with propargylated chalcones 40a-c and 41a-b under CuAAC conditions. 26 The reactions proceeded fairly in gently refluxing THF/H2O mixture containing L-ascorbic acid as a reducing agent and a catalytic amount of CuSO4•5H2O.

The chalcone modified cholesterol derivatives 46a, c, and 47b were active against E. coli. They were as active as ampicillin. Thus, the chalcones possessing unsubstituted phenyl, and p-dimethylaminophenyl, as well as 2thienyl alternatives were more active than other congeners against E. coli. The chalcone derivative 46c emerged as the most promising antimicrobial probe. It was as active as the controls against E. coli, S. aureus, and C. albicans.

In 2016, Liu et al. 27 described the synthesis of steroidal chalcones using the molecular hybridization approach.

The target molecules can be synthesized from the corresponding alkynes and steroidal azide 49 through click chemistry. The synthesis of the steroidal azide 49 is shown in Scheme 6. Bromoacetylation of DHEA under Schotten-Baumann conditions 28 afforded the corresponding bromide 1 in 87% yield, which then reacted with NaN3 in DMSO to give the steroidal azide 49 in 85% yield. Scheme 6. Synthesis of the steroidal azide 49.

The steroidal azide 49 was used directly for the next step after a simple aqueous work-up procedure. In the first step, a two-phase solvent system (DCM/H2O) was used; the base NaHCO3 in the aqueous phase neutralized the generated HBr, while the starting materials and the product were in the organic phase. Besides, this condition avoided unwanted side reactions of bromide 48 with H2O.

The synthesis of alkyne building blocks is shown in Scheme 7. Treatment of 4-hydroxy acetophenone and 4hydroxybenzaldehyde with propargyl bromide in the presence of K2CO3 gave compounds 50 and 51, respectively, which then underwent the KF/Al2O3-catalyzed Claisen-Schmidt condensations with aromatic aldehydes and acetophenones, affording substituted chalcones 52a-e and 53a-b. Scheme 7. Synthesis of alkyne building blocks 52 and 53.

The starting material was inexpensive pregnenolone acetate which was reduced by H2 to obtain compound 57. The intermediate 58 was acquired via a hydrolysis reaction. Subsequently, compound 58 was oxidized to yield the progesterone 59, whose acetyl group was then oxidized to generate the key intermediate 60. The compound 60 reacted with N-Boc-ethylenediamine to afford 61. The compound 61 was first deprotected and then reacted with various carboxylic acids to offer the final productions 62a-i (Scheme 9). The synthesis of target compounds 64 began from the intermediate 61 that was dehydrogenized by DDQ to form the intermediate 63.

Then, the final compounds 64a-i were obtained through deprotection and condensation reaction.

A preliminary biological activity evaluation was carried out. The antiproliferative results showed that most compounds exerted weak activity against human colon cancer cells HT-29 and lung cancer cells A549. In contrast, most compounds showed excellent growth inhibitory activity against human TNBC cells MDA-MB-231. Among these compounds, compounds 62c (with R1 = OMe and R2 = R3 = R4 = H) demonstrated the most potent activities with the IC50 values of 0.42 μM. Moreover, compound 62c could accumulate the ROS levels in MDA-MB-231 cells and induced cell apoptosis. Western Blot analysis documented that compound 62c could downregulate anti-apoptotic Bcl-2 but up-regulate pro-apoptotic Bax expression. Additionally, compound 62c could also activate the caspase-3 signaling pathway, which accelerated the apoptosis of MDA-MB-231 cells.

the low micromolar to the submicromolar range. Compound 72b with an IC50 value of 180 nM emerged as the most potent compound against rhDHFR. Interaction of the newly synthesized pregnenolone derivatives with hDHFR and estrogen receptor alpha (ERα) was also explored via docking simulations. The overall results of hDHFR inhibition have shown that these analogs can be further optimized and developed as potent anticancer agents.

Conclusions

The present review offers up-to-date literature on the syntheses of steroidal chalcones and derivatives reported during the last years. An almost infinite quantity of new molecules with various structures can be obtained by coupling two or more natural compounds. The objective of such syntheses is to combine the pharmacological properties of two or more biologically active molecules to increase or modulate these activities to obtain derivatives exhibiting new properties. In general, it has been observed that the unsaturated chalcone moiety in the synthesized compounds is at the origin of the activity of steroidal chalcones. Generally speaking, it is observed that steroids and the resulting derivatives continue to be of interest to researchers, given the biological potential inherent in this family of molecules.

©ARKAT USA, Inc Scheme 3. Synthesis of steroidal chalcones 22-37.

© ARKAT USA, Inc

Acknowledgements

The CNRS and the Ministère de l'Enseignement Supérieur et de la Recherche have financially supported this work.

Authors' Biographies