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Round scad mackerel sampled in 1995–1998 were analysed for genetic variation using mitochondrial-DNA and nuclear-DNA markers. Sequence variation for a fragment of the cytochromeb gene (mitochondrial), amplified by polymerase chain reaction, was screened across individuals using single strand conformation polymorphism (SSCP). Sequence analysis of all SSCP haplotypes indicated two mitochondrial clades separated by, on average, 2.3% nucleotide divergence. The geographic distribution of haplotypes was homogeneous (Weir and Cockerham's \( \hat \theta \) =–0.002). Also, no geographic heterogeneity was detected for length polymorphism of Intron 1 of the gene encoding aldolase B (\( \hat \theta \) =0.005). Although homogeneity in allele frequencies throughout the Indo-Malay archipelago conformed to the expectations for a widely distributed pelagic fish in a highly connected habitat, this was at variance with the sharp geographic structure previously uncovered in Indian scad mackerel, Decapterus russelli, a fish with life-history characteristics similar to D. macrosoma. A remarkable similarity, however, was the occurrence of two similarly distinct clades within each species, suggesting a common history of geographic isolation. Low sea levels in the Pleistocene might have caused the separation and vicariance of populations within both D. macrosoma and D. russelli. Subsequent genetic exchange between populations would then have erased allele-frequency differences at the cytochrome b and aldolase B loci in D. macrosoma while some barrier to gene flow was maintained in D. russelli.

Loubna Tazi a, Barry Kreiswirth b, Christian Carriere c, Michel Tibayrenc a,∗ a Genetics of Infectious Diseases, Unite mixte de Recherche Centre National de la Recherch Scientifique/Institut de Recherche pour le Developpement no. 9926, IRD, BP 64501, 34394 Montpellier Cedex 5, France b Public Health Research Institute Tuberculosis Center, International Center for Public Health, 225 Warren Street, Newark, NJ 07103, USA c Laboratoire de Bacteriologie, Hopital Arnaud de Villeneuve, 371 Avenue du Doyen Giraud, 34295 Montpellier Cedex 5, France

We evaluated cassava bacterial blight (CBB) infection in a pair-cross population of 150 individuals derived from an intra-specific cross between two non-inbred cassava (#Manihot esculenta$ Crantz) lines. The replicated trials were carried out in the field under high disease pressure over two consecutive crop cycles. Evaluations were conducted at 4 and 7 months after planting for the two cycles. Simple regression analysis and the nonparametric Kruskal-Wallis rank-sum test revealed that eight quantitative trait loci (QTLs) were invovled in resistance. We detected changes in QTLs from crop cycle to crop cycle. The pathogen population (#Xanthomonas axonopodis$ pv. #manihotis$) was also monitored over the period, using a restriction fragment length polymorphism probe and pathogenic tests. Changes in QTL detection over the 2 years could be correlated with changes in pathogen population structure. One QTL, located in linkage group D, was conserved over the two crop cycles, and in field to greenhouse evaluations. This study thus identified molecular markers useful for marker assisted-selection, a technique that can accelerate the long, multiple-season process of breeding for CBB resistance. (Résumé d'auteur)

Fifty-eight new isolates were obtained from root nodules of common bean (#Phaseolus vulgaris$) cultivated in soils originating from different agroecological areas in Senegal and Gambia (West Africa). A polyphasic approach including both phenotypic and genotypic techniques was used to study the diversity of the 58 #Rhizobium$ isolates and to determine their taxonomic relationships with reference strains. All the techniques performed, analysis of multilocus enzyme electrophoretic patterns, SDS-PAGE profiles of total cell proteins, PCR-RFLP analysis of the genes encoding 16S rRNA and of the 16S-23S RNA intergenic spacer region (ITS-PCR-RFLP), auxanographic tests using API galleries and nodulation tests lead to the consensus conclusion that the new rhizobial isolates formed two main distinct groups, I and II, belonging to #Rhizobium tropici$ type B and #Rhizobium etli$, respectively. By MLEE #R. etli$ and group II strains showed several related electrophoretic types, evidencing some extent of internal heterogeneity among them. This heterogeneity was confirmed by other techniques (ITS-PCR-RFLP, SDS-PAGE and host-plant-specificity) with the same nine distinct strains of group II showing some differences from the core of group II (54 strains). (Résumé d'auteur)

SDS-PAGE of total bacterial proteins was applied to the classification of 25 Sudanese and five Kenyan strains isolated from the root nodules of #Acacia senegal$ and #Prosopis chilensis$. Twenty strains were also studied by multilocus enzyme electrophoresis (MLEE) and the whole 16S rRNA gene was sequenced from two strains representing the two major clusters. These results, together with the previuously reported numerical taxonomy analysis, pulsed-field gel electrophoresis studies, DNA-DNA dot-blot hybridisation, genomic fingerprinting using repetitive sequence-basec PCR, DNA base composition analysis, DNA-DNA reassociation analysis, partial sequencing of the 16S rRNA gene and RFLP analysis of the amplified 16S rRNA gene, showed that all 30 strains belong to the genus #Sinorhizobium$. Two of the strains grouped with #Sinorhizobium saheli$ and seven with #Sinorhizobium terangae$, while the rest did not cluster with any of the established species. The majority of the strains formed two phenotypically and genotypically distinct groups and we therefore propose that these strains should be classified as two new species, #Sinorhizobium arboris$ sp. nov. and #Sinorhizobium kostiense$ sp. nov. (Résumé d'auteur)

Eight Frankia-Gymnostoma nitrogen-fixing symbioses endemic to New Caledonia were studied using a range of techniques including molecular typing of Frankia , infectivity/effectivity and host spectrum of the isolates. The study was conducted on 128 Frankia isolates from the eight Gymnostoma species. The RFLP analysis of the rRNA 16S-23S ITS allowed us to cluster the strains in four groups. Symbiotic characterization of the four groups failed to reveal any host specificity both in term of infectivity or effectivity. The characteristics of the strains were those of Eleagnaceae -infective Frankia strains. Co-inoculation of two Gymnostoma species with mixtures of strains evidenced 11 % of mixed RFLP profiles. In such controlled conditions, this was interpreted as co-infections. This work evidenced original features of these Gymnostoma isolates: (i) they were closer to Eleagnaceae -infective Frankia than Casuarina -infective Frankia ; (ii) closer to atypical Frankia from Casuarina ; (iii) poorly host-specific; and (iv) they had a tendency of co-infection. They could represent a primitive group of Frankia able to survive in a wide range of habitats.

The restriction fragment length polymorphism of the 195 bp repeated DNA sequence of #Trypanosoma cruzi$ was analyzed among 23 #T. cruzi$ stocks giving a reliable picture of the whole phylogenetic variability of the species. The profiles observed with the enzymes Hinf1 and Hae3 were linked together and supported the existence of two groups. Group 1 shows a 195 bp repeated unit (Hinf 1) and high molecular weight DNA (Hae 3), while group 2 presents a ladder profile for each enzyme, which is a characteristic of tandemly repeated DNA. The two groups, respectively, clustered stocks pertaining to the two principal lineages evidenced by izoenzyme and RAPD markers. The congruence among these three independent genomic markers corroborates the existence of two real phylogenetic lineages in #T. cruzi$. The specific monomorphic profiles for each major phylogenetic lineage suggest the existence of ancient sexuality and cryptic biological speciation. (Résumé d'auteur)

Polyploids in Tripsacum, a wild relative of maize, reproduce through the diplosporous type of apomixis, an asexual mode of reproduction through seeds. Diplosporous apomixis involves both the failure of meiosis and the parthenogenetic development of the unreduced gametes, resulting in progenies that are exact genetic copies of the mother plant. Apomixis is believed to be controlled by one single dominant allele, responsible for the whole developmental process. Construction of a linkage map for the chromosome controlling diplosporous apomixis in Tripsacum was carried out in both tetraploid-apomictic and diploid-sexual Tripsacum species using maize restriction fragment length polymorphism (RFLP) probes. A high level of collinearity was observed between the Tripsacum chromosome carrying the control of apomixis and a duplicated segment in the maize genome. In the apomictic tetraploid, there was a strong restriction to recombination, as compared to the corresponding genomic segment in sexual plants and maize. This suggests that apomixis, although inherited as a single Mendelian allele, might really be controlled by a cluster of linked loci. The analysis also revealed the tetrasomic nature of the inheritance of the chromosomal segment controlling apomixis, which contradicts the usually accepted hypothesis of an allopolyploid origin of apomictic species. The implications of these data for the transfer of apomixis into cultivated crops are discussed, and a new approach to studying the genetics of apomixis, based on comparative mapping, is proposed.

The Anopheles genus is probably one of the best studied genera among insects of medical importance. Of more than 500 species currently listed in the world about sixty species are vectors of malaria agents and about thirty species are responsible for most of the transmission [1-3]. The important epidemiological role of anopheline species has motivated many studies of taxonomy and systematics with traditional tools. With the advent of molecular tools, the development of informatics databases and new mathematical concepts on shape, characteriz‐ ing insects has become more and more accurate. Molecular tools based on the nucleotide polymorphism of DNA have allowed the identification of cryptic diversity, confirming and refining previous findings suggesting the existence of many species groups or complexes of sibling species. The development of international genetic sequence database collabora‐ tionshttp://www.insdc.org, http://www.barcodinglife.com/ allowed the use of reference sequences for species identification. Although not well developed, the same need for infor‐ matic databases arose for traditional taxonomy.

The risk of serious infections caused by Staphylococcus aureus is well-known. However, most studies regarding the distribution of (clinically relevant) S. aureus among humans and animals took place in the western hemisphere and only limited data are available from (Central) Africa. In this context, recent studies focused on S. aureus strains in humans and primates, but the question of whether humans and monkeys share related S. aureus strains or may interchange strains remained largely unsolved. In this study we aimed to evaluate the distribution and spread of human-like S. aureus strains among great apes living in captivity. Therefore, a primate facility at the International Centre for Medical Research of Franceville (Gabon) was screened. We detected among the primates a common human S. aureus strain, belonging to the spa -type t148. It was isolated from three different individuals of the western lowland gorilla ( Gorilla gorilla gorilla ), of which one individual showed a large necrotizing wound. This animal died, most probably of a staphylococcal sepsis. Additionally, we discovered the t148 type among chimpanzees ( Pan troglodytes ) that were settled in the immediate neighbourhood of the infected gorillas. A detailed analysis by pulsed field gel electrophoresis showed that the gorilla and chimpanzee isolates represented two closely related strains. To our knowledge, this is the first report of a human-associated S. aureus strain causing disease in great apes. The simultaneous detection in gorillas and chimpanzees indicated an interspecies transmission of this S. aureus strain. Our results recommend that protection of wild animals must not only be based on habitat conservation, but also on the assessment of the risk of contact with human pathogens.

Ninety-three accessions representing 21 species from the genus Oryza were examined for restriction fragment length polymorphism. The majority (78%) of the accessions, for which five individuals were tested, were found to be monomorphic. Most of the polymorphic accessions segregated for only one or two probes and appeared to be mixed pure lines. For most of the Oryza species tested, the majority of the genetic variation (83%) was found between accessions from different species with only 17% between accessions within species. Tetraploid species were found to have, on average, nearly 50% more alleles (unique fragments) per individual than diploid species reflecting the allopolyploid nature of their genomes.Classification of Oryza species based on RFLPs matches remarkably well previous classifications based on morphology, hybridization and isozymes. In the current study, four species complexes could be identified corresponding to those proposed by Vaughan (1989): the O. ridleyi complex, the O. meyeriana complex, the O. officinalis complex and the O. sativa complex. Within the O. sativa complex, accessions of O. rufipogon from Asia (including O. nivara) and perennial forms of O. rufipogon from Australia clustered together with accessions of cultivated rice O. sativa. Surprisingly, indica and japonica (the two major subspecies of cultivated rice) showed closer affinity with different accessions of wild O. Rufipogon than to each other, supporting a hypothesis of independent domestication events for these two types of rice. Australian annual wild rice O. meridionalis (previously classified as O. rufipogon) was clearly distinct from all other O. rufipogon accessions supporting its recent reclassification as O. meridionalis (Ng et al. 1981). Using genetic relatedness as a criterion, it was possible to identify the closest living diploid relatives of the currently known tetraploid rice species. Results from these analyses suggest that BBCC tetraploids (O. malampuzhaensis, O. punctata and O. minuta) are either of independent origins or have experienced introgression from sympatric C-genome diploid rice species. CCDD tetraploid species from America (O. latifolia, O. alta and O. grandiglumis) may be of ancient origin since they show a closer affinity to each other than to any known diploid species. Their closest living diploid relatives belong to C genome (O. eichingeri) and E genome (O. Australiensis) species. Comparisons among African, Australian and Asian rice species suggest that Oryza species in Africa and Australia are of polyphyletic origin and probably migrated to these regions at different times in the past.Finally, on a practical note, the majority of probes used in this study detected polymorphism between cultivated rice and its wild relatives. Hence, RFLP markers and maps based on such markers are likely to be very useful in monitoring and aiding introgression of genes from wild rice into modern cultivars.

BackgroundAnopheles gambiae, a major vector of malaria, is widely distributed throughout sub-Saharan Africa. In an attempt to eliminate infective mosquitoes, researchers are trying to develop transgenic strains that are refractory to the Plasmodium parasite. Before any release of transgenic mosquitoes can be envisaged, we need an accurate picture of the differentiation between the two molecular forms of An. gambiae, termed M and S, which are of uncertain taxonomic status.Methodology/principal findingsInsertion patterns of three transposable elements (TEs) were determined in populations from Benin, Burkina Faso, Cameroon, Ghana, Ivory Coast, Madagascar, Mali, Mozambique, Niger, and Tanzania, using Transposon Display, a TE-anchored strategy based on Amplified Fragment Length Polymorphism. The results reveal a clear differentiation between the M and S forms, whatever their geographical origin, suggesting an incipient speciation process.Conclusions/significanceAny attempt to control the transmission of malaria by An. gambiae using either conventional or novel technologies must take the M/S genetic differentiation into account. In addition, we localized three TE insertion sites that were present either in every individual or at a high frequency in the M molecular form. These sites were found to be located outside the chromosomal regions that are suspected of involvement in the speciation event between the two forms. This suggests that these chromosomal regions are either larger than previously thought, or there are additional differentiated genomic regions interspersed with undifferentiated regions.

A thermophilic terephthalate-degrading methanogenic consortium was successfully enriched for 272 days in an anaerobic hybrid reactor, and the microbial structure was characterized using terminal RFLPs, clone libraries and fluorescence in-situ hybridization with rRNA-targeted oligonucleotide probes. All the results suggested that Methanothrix thermophila-related methanogens, Desulfotomaculum-related bacterial populations in the Gram-positive low-G + C group, and OP5-related populations were the key members responsible for terephthalate degradation under thermophilic methanogenic conditions except during periods when the reactor experienced heat shock and pump failure. These perturbations caused a significant shift in bacterial population structure in sludge samples taken from the sludge bed but not from the surface of the packing materials. After system recovery, many other bacterial populations emerged, which belonged mainly to the Gram-positive low-G + C group and Cytophaga-Flexibacter-Bacteroides, as well as beta-Proteobacteria, Planctomycetes and Nitrospira. These newly emerged populations were probably also capable of degrading terephthalate in the hybrid system, but were out-competed by those bacterial populations before perturbations.

www.springer-ny.com; International audience; In order to promote genome research on coffee trees, one of the most important tropical crops, a bacterial artificial chromosome (BAC) library of the coffee allotetraploid species, Coffea arabica, was constructed. The variety IAPAR 59, which is widely distributed in Latin America and exhibits a fair level of resistance to several pathogens, was chosen. High-efficiency BAC cloning of the high molecular weight genomic DNA partially digested by HindIII was achieved. In total, the library contains 88,813 clones with an average insert size of 130 kb, and represents approximately eight C. arabica dihaploid genome equivalents. One original feature of this library is that it can be divided into four sublibraries with mean insert sizes of 96, 130, 183 and 210 kb. Characterisation of the library showed that less than 4.5% of the clones contained organelle DNA. Furthermore, this library is representative and shows good genome coverage, as established by hybridisation screening of high-density filters using a number of nuclear probes distributed across the allotetraploid genome. This Arabica BAC library, the first large-insert DNA library so far constructed for the genus Coffea, is well-suited for many applications in genome research, including physical mapping, map-based cloning, functional and comparative genomics as well as polyploid genome analyses.

The diversity of Bradyrhizobium strains nodulating three cowpea (Vigna unguiculata L. Walp.) cultivars in favorable and water-limited conditions occuring at flowering was analysed. PCR- Restriction Fragment Length Polymorphism (PCR-RFLP) analysis of 16S-23S rDNA intergenic spacer region (IGS) directly applied on 85 crushed nodules distinguished four genetic profiles, IGS types I, II, III and IV. The distribution of these IGS types according to water conditions and cowpea cultivars (B-21, TN 88-63 and Mouride) showed that nodulating strains appeared more diverse in water-limited condition. More than three quarters of prospected nodules presented the IGS type I. They were formed on all three cultivars and in both water conditions. Only a small part of nodules was distributed between the IGS type II, III and IV. Nodules showing the IGS types II and III were found mainly in limited conditions on TN 88-63 and Mouride cultivars, whereas nodules presenting the IGS type IV were collected only from cultivars B-21 and Mouride, in both water conditions. Strains corresponding to the different profiles were isolated. The phylogenetic analysis of 16S rRNA gene sequences showed that they belong to the genus, Bradyrhizobium. The sequence analysis of 16S-23S rDNA IGS revealed that the strains exhibiting IGS types II, III and IV were closely related to some Faidherbia albida isolates from Senegal. IGS type II can be assigned with at least 98% similarity to Bradyrhizobium genospecies IV. IGS types III and IV showed more than 96% similarity with genospecies VII and could belong to the same genospecies. IGS type I, the most frequent, exhibits low IGS similarity with reported sequences in the databases, and could represent a new genospecies. (African Journal of Biotechnology: 2003 2(1): 13-23)

Coffea canephora# possede l'une des aires de repartition les plus larges du sous-genre #Coffea# : elle s'etend d'ouest en est, de la Guinee au Soudan et, du nord au sud, du Cameroun a l'Angola. La conservation a long terme de cette espece s'effectue en champs a cause du comportement peu orthodoxe de ses graines lors de leur stockage. La premiere analyse de la diversite genetique de cette espece a ete realisee a partir du polymorphisme enzymatique. L'utilisation des marqueurs moleculaires est recente et les resultats presentes dans ce chapitre constituent les premieres donnees sur la variabilite moleculaire de #C. canephora#. L'analyse de la diversite agromorphologique ne permet pas de reveler une forte structuration de l'espece. A l'aide des locus isoenzymatiques discriminant les groupes guineen et congolais, un schema de selection recurrente reciproque de #C. canephora# fonde sur l'utilisation de ces deux groupes a ete propose et son efficacite a ete validee par la suite, ce qui confirme qu'il est important de connaitre la structure de la diversite pour exploiter au mieux les ressources genetiques. Les resultats de l'etude sur les marqueurs moleculaires devraient permettre d'ameliorer ce schema de selection. Un certain nombre de recommandations pour assurer une meilleure gestion #ex situ# des ressources genetiques est avance

Rice progenies used for the construction of genetic maps permit exhaustive identification and characterization of resistance genes present in their parental cultivars. We inoculated a rice progeny derived from the cross IR64 x Azucena with different Magnaporthe grisea isolates that showed differential responses on the parental cultivars. By QTL mapping, nine unlinked loci conferring resistance to each isolate were identified and named Pi-24( t) to Pi-32( t). They could correspond to nine specific resistance genes. Five of these resistance loci (RLs) were mapped at chromosomal locations where no resistance gene was previously reported, defining new resistance genes. Using degenerate primers of the NBS (nucleotide binding site) motif found in many resistance genes, two resistance gene analogues (RGAs) IR86 and IR14 were identified and mapped closely to two blast RLs (resistance identified in this study, i.e. Pi-29(t) and Pi-30(t) respectively). These two RLs may correspond to the Pi-11 and Pi-a blast resistance genes previously identified. Moreover, the ir86 and ir14 genes have been identified "in silico" on the indica rice cultivar 93-11, recently sequenced by Chinese researchers. Both genes encodes NBS-LRR-like proteins that are characteristics of plant-disease resistance genes.

Microsatellites or simple sequence repeats (SSR) were used to assess polymorphism among 16 Coffea arabica and four Coffea canephora accessions, and to identify DNA introgression fragments from C. canephora in four C. arabica lines. Thirty-one primer pairs allowed for the identification of 92 polymorphic alleles distributed over 37 loci. The C. arabica accessions derived from the genetic bases 'Typica' and 'Bourbon' were grouped separately according to their genetic origin. Two genotypes derived from a spontaneous hybrid (C. arabica x C. canephora) were classified with the C. canephora accessions from Central Africa. Coffea canephora from West Africa were separated from the other accessions studied. Four alleles related to introgression (i.e. present in C. canephora and introgressed lines, and absent in C. arabica) were identified. The SSR markers were used successfully for characterization of a particular cultivar ('Veranero') from Costa Rica, which is known for its late maturity. (Resume d'auteur)

A direct comparison of the genetic linkage maps of sorghum and rice is proposed. It is based on the mapping of a common set of 123 RFLP probes scattered on the genomes of both species. For each species a composite map was established by merging two individual maps comprising many common loci. This enabled us to confirm the global correspondence scheme that had previously been established between the chromosomes of sorghum and rice. It also provided a more detailed insight into the conservation of synteny and colinearity : 69% of the loci mapped on a given rice chromosome mapped to the corresponding homoeologous chromosome in sorghum ; among them, 84% formed a colinear arrangement between the two species. Local inversions and translocations were detected. (Résumé d'auteur)

Pineapple, Ananas comosus (L.) Merr, is the third most important tropical fruit cultivated in all tropical and subtropical countries. Pineapple germplasm includes all seven species of the genus Ananas and the unique species of the related genus Pseudananas. A knowledge of its diversity structure is needed to develop new breeding programs. Restriction fragment length polymorphism (RFLP) was used to study molecular diversity in a set of 301 accessions, most of which were recently collected. This sample was analysed using 18 homologous genomic probes. Dissimilarities were calculated by a Dice index and submitted to Factorial Analysis. The same data were represented as a diversity tree constructed with the score method. Pseudananas sagenarius displayed a high polymorphism and shares 58.7% of its bands with Ananas. Within Ananas, variation appears continuous and was found mostly at the intraspecific level, particularly in the wild species Ananas ananassoides and Ananas parguazensis. As for the cultivated species, Ananas comosus appears relatively homogeneous despite its wide morphological variation and Ananas bracteatus, which is grown as a fence and for fruit, appears still much less variable. By contrast Ananas lucidus, cultivated by the Amerindians for fiber, displays a high polymorphism. This tree displayed a loose assemblage of numerous clusters separated by short distances. Most species were scattered in various clusters, a few of these being monospecific. Some accessions which had not been classified, as they shared morphological traits typical of different species, re-group with one or the other, and sometimes with both species in mixed clusters. No reproductive barrier exists in this germplasm and these data indicate the existence of gene flow, enhancing the role of effective sexual reproduction in a species with largely predominant vegetative mutiplication.

Cassava bacterial blight (CBB), caused by #Xanthomonas axonopodis$ pv. #manihotis$ (#Xam$), is a major disease of cassava (#manihot esculenta$ Crantz) in Africa and South America. Planting resistant varieties is the preferred method of disease control. Recent genetic mapping of an F1 cross (TMS 30572 X CM 2177-2) led to the construction of the first molecular genetic map of cassava. To better understand the genetics of resistance to CBB, we evaluated individuals of the F1 cross for CBB resistance by controlled greenhouse inoculations and visually assessed symptoms on days 7, 15, and 30 days after inoculation, using a scale where 0 = no disease and 5 = maximum susceptibility. Five #Xam$ strains were used : CIO-84, CIO-1, CIO-136, CIO-295 and ORST X-27. Area under the disease progress curve (AUDPC) was used as a quantitative measure of resistance in QTL analysis by single-marker regression. Based on the AUDPC values, eight QTLs (Quantitative trait loci), located on linkage groups B, D, L, N, and X of the female-derived framework map, were found to explain 9-20% of the phenotypic variance of the crop's response to the #Xam$ strains. With the male-derived framework map, four QTLs on linkage groups G and C explained 10.27-27.1% of the variance. A scheme to confirm the usefulness of these markers in evaluating segregating populations for resistance to CBB is proposed. (Résumé d'auteur)

We examined the genotypic diversity of 64 Bradyrhizobium strains isolated from nodules from 27 native leguminous plant species in Senegal (West Africa) belonging to the genera Abrus , Alysicarpus , Bryaspis , Chamaecrista , Cassia , Crotalaria , Desmodium , Eriosema , Indigofera , Moghania , Rhynchosia , Sesbania , Tephrosia , and Zornia , which play an ecological role and have agronomic potential in arid regions. The strains were characterized by intergenic spacer (between 16S and 23S rRNA genes) PCR and restriction fragment length polymorphism (IGS PCR-RFLP) and amplified fragment length polymorphism (AFLP) fingerprinting analyses. Fifty-three reference strains of the different Bradyrhizobium species and described groups were included for comparison. The strains were diverse and formed 27 groups by AFLP and 16 groups by IGS PCR-RFLP. The sizes of the IGS PCR products from the Bradyrhizobium strains that were studied varied from 780 to 1,038 bp and were correlated with the IGS PCR-RFLP results. The grouping of strains was consistent by the three methods AFLP, IGS PCR-RFLP, and previously reported 16S amplified ribosomal DNA restriction analysis. For investigating the whole genome, AFLP was the most discriminative technique, thus being of particular interest for future taxonomic studies in Bradyrhizobium , for which DNA is difficult to obtain in quantity and quality to perform extensive DNA:DNA hybridizations.

An interspecific partial genetic linkage map of #Coffea$ sp. based on 62 backcross hybrids is presented. F1 hybrids were generated by a cross between the wild #C. pseudozanguebariae$ and the anciently cultivated #C. liberica$ var. #dewevrei$ (DEW) ; progeny were then derived from a backcross between F1 hybrid and DEW. The map construction consisted of a two-step strategy using 5.5 and 3.1 LOD scores revealed by simulation file. The map consisted of 181 loci : 167 amplified fragment length polymorphism (AFLP) and 13 radom fragment length polymorphism (RFLP) loci. The markers were assembled into 14 linkage groups, each with 4-31 markers covering 1,144 cM. Segregation distorsion was observed for 30% of all loci, in particular 3:1 and 1:3 ratios equally favouring each of the two parents. The existence of such ratios suggests genetic conversions events. This map also represents an initial step towards the detection of quantitative trait loci. (Résumé d'auteur)

The technique described here is a fast and simple method of extracting chloroplast DNA (cpDNA). It overcomes the need for differential centrifugation using density gradients. The leaves do not have to be kept in the dark and lyophilized before extraction, but lyophilization is still possible. The chloroplasts are specifically lysed in a cell extract of leaves, using a non-ionic detergent. After isolation by centrifugation, the cpDNA is purified by the combined action of proteolytic enzymes and detergents, followed by the elimination of proteins using a mixture of chloroform and isoamyl alcohol. This method provided good quality restriction profiles for all species analyzed.

At the present time, the precise taxonomial status of many strains classified as #Bradyrhizobium$ still remains unclear. There is a need to develop a reliable grouping method, specifying the genetic relationships between these strains. Phenotypic methods as auxanography or protein profiling did not prove valuable for classification of bradyrhizobia and were not in good agreement with phylogenetic data. The purpose of this work is to develop a strategy to analyse the diversity of bradyrhizobia and to identify genomic groups among our collection of strains isolated from 9 small legume species in Senegal. #B. japonicum$, #B. elkanii$ and representatives of previously described groups by Moreira et al. (1993) and Dupuy et al. (1994) were included as references. Bacterial diversity was assessed by two different techniques, PCR-RFLP analysis of the IGS region between 16S and 23S rDNA (IGS) and the AFLP technique. Groupings of strains obtained by the two methodologies will be presented and compared. 16S rDNA PCR-RFLP analysis of strains representative of IGS clusters from small legumes was also performed. (Résumé d'auteur)

One of the enduring problems in the epidemiology of sleeping sickness is that there are 3 morphologically indistinguishable subspecies of Trypanosoma brucei involved in a complex transmission cycle between humans, tsetse and reservoir hosts. Two subspecies, T. b. gambiense and T. b. rhodesiense, are infective to man and cause gambian and rhodesian sleeping sickness, respectively. The third subspecies T. b. brucei cannot by definition infect humans, but coexists with the other trypanosomes in reservoir hosts and vectors.

La NouvelleCalédonie possède une forte concentration en Casuarinaceae endémiques. On y dénombre huit espèces de #Gymnostoma# et une espèce de #Casuarina : C. collina#. Vingt-six souches de #Frankia# ont été isolées de nodules de #C. collina# et étudiées par diverses techniques incluant l'infectivité, l'effectivité, le spectre d'hôte et la PCR/RFLP de l'ITS 16S-23S. Tous ces #Frankia# présentent les mêmes caractéristiques que celles décrites chez les souches atypiques de #Casuarina# spp. Ce travail a mis en évidence, chez les #Frankia# de #C. collina#, les caractéristiques originales suivantes : (i) ils sont localisés à l'intérieur des nodules ; (ii) ils appartiennent au groupe des #Frankia# infectifs sur #Elaeagnus#;(iii) ils se regroupent dans quatre groupes ITS déjà décrits chez #Gymnostoma#. En fait, il est très probable que les souches atypiques de #C. collina# et les #Frankia# de #Gymnostoma# soient les mêmes. On peut formuler l'hypothèse que les #Frankia# de #Gymnostoma# pourraient utiliser #C. collina# comme alternative en absence de leur hôte naturel ou quand les conditions écologiques sont favorables à #C. collina#.

Analyses of DNA polymorphism and virulence variation were used to evaluate the population structure of Xanthomonas axonopodis pv. manihotis, the pathogen causing cassava bacterial blight in Colombia. We collected strains from the major cassava-growing regions which can be grouped into different edaphoclimatic zones (ECZs) according to environmental conditions, production constraints, and economic parameters. DNA polymorphism was assessed by a restriction fragment length polymorphism analysis, using an X. axonopodis pv. manihotis plasmid DNA sequence (pthB) as a probe to evaluate the genetic relatedness among 189 Colombian strains. The sampling intensity permitted the estimation of genetic differentiation within and among ECZs, sites, and fields and even within an individual plant. A multiple correspondence analysis indicated that the Colombian X. axonopodis pv. manihotis population showed a high degree of diversity relative to X. axonopodis pv. manihotis populations studied previously, and the entire collection was grouped into seven clusters. A general correlation was observed between the clusters and the geographical origin of the strains, as each cluster was largely composed of strains from the same ECZ. Representative strains, identified with pthB, were further characterized by ribotyping, hybridization to two repetitive genomic probes (pBS6 and pBS8), and restriction analysis of plasmid contents to evaluate the complementarity of these markers. Virulence variation was observed within the Colombian collection. Strains of different aggressiveness were found in all ecological zones, but no correlation between virulence variation and DNA polymorphism was observed. The genetic and virulence analyses contribute to understanding the X. axonopodis pv. manihotis population structure in Colombia.

L'analyse du polymorphisme de longueur des fragments de restriction d'ADN amplifie (PCR-RFLP) presente de nombreux avantages pour la caracterisation de la diversite microbienne: relative rapidite, faible cout, possibilite d'application a l'ensemlble ries taxons bacteriens, bonne reproductibilite, possibilite d'analyse simultanee d'un grand nombre de souches, interet au niveau taxonomique. Afin d'optimiser sort usage, nous avons cherche a determiner combien d'enzymes et lesquels etaient le plus approprie, en effectuant une analyse systematique par ordinateur de la capacite de 25 enzymes de restriction differentes a generer par PCR-RFLP, seules ou en combinaison, (les profils de digestion permettant la distinction d'especes proches taxonomiquement. La sous-unite 16S de l'ARN ribosomal (rrs; pour ribosomal RNA small subunit) a ete choisie pour simuler ces PCR-RFLP: elle correspond a la region la plus frequemment etudiee et permet en general une bonne discrimination au niveau de l'espece; de plus, de nombreuses sequences rrs sont disponibles dans les banques de donnees. L'etude a ete focalisee sur les rhizobia qui representent un bon modele de genres differents, entremeles avec d'autres genres tres proches taxonomiquement, et parce que leur importance en agriculture induit la necessite d'outils performants pour la caracterisation de grands nombres de nouveaux isolats. La digestion de 24 sequences rrs correspondant aux souches-types de differentes especes a ete simulee par ordinateur. Les profils de digestion obtenus pour les differentes sequences ont ete compares deux par deux afin de determiner le nombre de profils distincts generes par chaque enzyme individuellement. ou par les combinaisons systematiques de 2, 3, 4, 5, 6 ou 7 enzymes. Differentes combinaisons de 3, 4 ou 5 enzymes permettent la distinction entre les especes. La frequence a laquelle les differentes enzymes sont trouvees dans les i:ounfiinaisons discrimimantes ne correspond pas completement a leur pouvoir de discrimation individuel, mais a la complementarite qu'elles presentent lorsqu'ellas sont analysees ensemble. La combination des profils obtenus avec six enzymes, choisies parmi les sept puis frequentes dans les combinaisons discriminantes, permet en moyenne de distinguer plus de 99,5%, des sequences etudiees, quelle que soit la similarite entre ces sequences. L'utilisation d'au moins six de ces sept enzymes est suggeree afin d'assurer une bonne distinction des especes de rhizobia, connues et inconnues.

The biodiversity of nitrogen-fixing organisms is huge. Taxonomic and phylogenetic research is needed to structure this diversity, to facilitate communication among scientists, and to increase our understanding of the evolution and biology of diazotrophs. Molecular tools for taxonomic and biodiversity studies of diazotrophic rhizobia, frankiae, cyanobacteria and bacilli are presented in sections 2 to 5. Sections 6 to 9 focus on problems with genus and species assignment.

Neptunia oleracea is an annual aquatic stem-nodulated legume indigeneous of waterlogged places in Senegal. Bacterial isolates from nodules of N. oleracea can infect Medicago sativa roots but the nodules are not efficient. No information is available on the taxonomy of Neptunia specific bacteria.

We analysed the genetic differentiation among 17 natural populations of the Nile tilapia Oreochromis niloticus (Linnaeus, 1758) using allozymes and restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA). The populations studied, from the River Senegal to Lake Tana and from Lake Manzalla to Lake Baringo, represent all subspecies which have been previously described. Sixteen variable nuclear loci showed that these populations can be clustered in three groups: (1) West African populations (Senegal, Niger, Volta and Chad drainages), (2) Ethiopian Rift Valley populations (Lakes Awasa, Ziway, Koka and the Awash River) and (3) Nile drainage (Manzalla, Cairo, Lake Edward) and Kenyan Rift Valley populations (Lakes Turkana, Baringo and River Suguta). Nine different mtDNA haplotypes were found in the RFLP analysis of a 1 kb portion of the D-loop region. The network obtained showed that there are three geographically distinct groups; all West African populations and O. aureus are clustered, the two Ethiopian Rift Valley populations are distinct and between these two groups are the Kenyan and Ugandan Rift Valley populations. Nile populations show affinities both with West African populations and with specimens from Lakes Tana and Turkana. Taxonomic and biogeographical implications of these results are discussed.

Two doubled-haploid rice populations, IR64/Azucena and IRAT177/Apura, were used to identify markers linked to rice yellow mottle virus (RYMV) resistance using core restriction fragment length polymorphism (RFLP) maps. Resistance was measured by mechanical inoculation of 19-day-old seedlings followed by assessment of virus content by enzyme-linked immunosorbent assay tests 15 days after inoculation. IR64/Azucena and IRAT177/Apura populations, 72 and 43 lines, respectively, were evaluated, and resistance was found to be polygenic. Resistance was expressed as a slower virus multiplication, low symptom expression, and limited yield loss when assessed at the field level. Bulked segregant analysis using the IR64/Azucena population identified a single random amplified polymorphic DNA marker that mapped on chromosome 12 and corresponded to a major quantitative trait locus (QTL) evidenced by interval mapping. When pooling RFLP data, integrated mapping of this chromosome revealed that the QTL was common to the two populations and corresponded to a small chromosomal segment known to contain a cluster of major blast resistance genes. This region of the genome also reflected the differentiation observed at the RFLP level between the subspecies #indica$ and #japonica$ of #Oryza sativa$. This is consistent with the observation that most sources of RYMV resistance used in rice breeding are found in upland rice varieties that typically belong to the #japonica$ subspecies. (Résumé d'auteur)

A sample of African #Clarias$ catfishes from the Senegal River was studied using morphometry, allozyme variation, microsatellites and RFLPs of mitochondrial DNA. They all confirmed the presence of two species, #C. gariepinus$ and #C. anguillaris$. The two species were closely related genetically and no diagnostic loci were found in allozymes and microsatellites studies. Two of the 11 haplotypes of mtDNA observed were shared by both species. Three of the four assays (morphometry, allozymes and microsatellites) allowed a precise characterization of both. One specimen occupied an intermediate position in the analysis of the data ; it was considered an F1 hybrid whose possible origin is discussed. (Résumé d'auteur)

From 1989 to 1992, a longitudinal study of the relationships between different tick species and domestic ungulates in the transmission and amplification of Crimean-Congo hemorrhagic fever (CCHF) virus was undertaken in the Bandia area in Senegal where the presence of the virus had been reported previously. An epizootic occurred in 1991-1992 and 22 strains of CCHF virus were isolated from Hyalomma marginatum rufipes Koch, Amblyomma variegatum (F.), Rhipicephalus guilhoni Morel & Vassiliades, and R. evertsi evertsi Neumann ticks collected from cattle and goats. No human cases were reported. Transmission of CCHF virus in the area involves a complicated pattern including many tick species and hosts. Amplicons of the S fragment (536 bp) of the CCHF genome of 12 isolates from the study were obtained by polymerase chain reaction and analyzed by restriction-length fragment polymorphism. Three different genotypes of CCHF virus were identified and present during the epizootic. One genotype was recovered from A. variegatum, R. guilhoni, and R. e. evertsi and 2 genotypes were isolated from H. m. rufipes.

Cultivated varieties of rice are the result of several thousands of years of human selection from the available genetic diversity, in various natural environments and human cultures. The process of gathering varieties from different geographic origins, planting them in the same field, and selecting particular genotypes among the offsprings of natural hybridization can still be observed in traditional societies and has no doubt been going on since early days. Modern breeding in the last century has done little more than to control the process of hybridization and selection in a more efficient way, and the result has been to adapt varieties to better controlled and fertilized environments. The basic approach consists of choosing parents with complementary characters in order to combine them. Unexpected (transgressive) characters are also obtained in the process. Evaluation of germplasm is generally a matter of screening for useful characters although the understanding of the genetic structure of genetic resources can give a clue to obtain transgressive characters (Second and Charrier 1989).

Plasmids were recovered by an alkaline procedure from six symbiotically effective strains of Rhizobium meliloti of diverse geographical origin, reported to harbour only one middle-size large plasmid (ranging from 89 to 143 Megadaltons). Each purified plasmid was digested with eight restriction endonucleases; cleavage patterns were very complex: only KpnI and XbaI gave a limited number of bands. Fingerprints were very different, whatever the restriction enzyme or the geographical origin of the strains. However, Southern DNA-DNA hybridizations revealed that the plasmids showed homologous sequences having a high thermal stability. We gave evidence that some of these sequences are common to all the plasmids of R. meliloti. The biological function of these common sequences is unknown. Hybridization with cloned nitrogen fixation (nif) genes from Klebsiella pneumoniae had demonstrated that nif genes were not located on the middle -- size plasmids of R. meliloti studied in this paper.

Fifty-one fast growing rhizobial strains isolated from root nodules of #Acacia senegal$ and #Prosopis chilensis$ in Sudan and Kenya were divided into DNA homology groups using non-radioactive DNA-DNA dot-blot hybridisation. #Rhizobium leguminosarum$ , #R. galegae$, #R. tropici$, #Mesorhizobium loti$, #Sinorhizobium fredii$, #S. meliloti$ used in numerical taxonomy were included in the hybridisation experiments as reference strains and, at a later strage #S. saheli$ and #S. terangae$. Scores given to the intensities of dots detected in the hybridisation experiments were used in principal component analysis, which clustered the majority of the tree rhizobia in two separate DNA-homology groups. The 51 strains were also analysed by genomic fingerprinting using the repetitive sequence-based polymerase chain reaction (rep-PCR) method with REP, ERIC, BOX and GTG5 primers. The resulting genomic fingerprints were compared with strains representing 15 rhizobial species. The relationship of 17 Sudanese strains to established sinorhizobial species was examined using the optical renaturation rates method and the G+C content of nine strains was determined. Results from dot-blot hybridisation and rep-PCR experiments were found to be in close agreement with each other and with the results obtained from spectrophotometric reassociation analysis. We suggest that rep-PCR fingerprinting can be used as a first and dot-blot hybridisation as a second rapid and dependable genomic screening method to classify new rhizobial isolates of unknown taxonomic status and to choose the representative strains for the more laborious DNA-DNA reassociation experiments. (Résumé d'auteur)

The African Anopheles funestus and the Asian An. minimus groups are closely related and are probably considered distinct only because of their geographic separation. This study aimed at improving two identification methods based on polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) already devel- oped for either group. Each PCR-RFLP, either on the internal transcribed spacer 2 (ITS2) for the An. minimus group, and domain 3 (D3) for the An. funestus group, was applied to the other group for the standardization of one identifi- cation method applicable on both continents. The ITS2 fragment digested by Bsi ZI showed the highest diagnostic power. This assay allowed the discrimination of at least 13 Anopheles species within the subgenus Cellia from two continents (Africa and Asia), among which are five major malaria vectors. Moreover, digestion of the D3 with Msp I showed intragenomic variations within An. funestus populations. Two types of D3 copies (M and W) occurred in specimens from southern Africa. The populations from West-Central Africa presented only type W and East-Malagasy populations exhibited type M. Since An. funestus shows a great capacity of adaptation, these molecular variations, along with behavioral and ecologic ones, reinforce the hypothesis of a species complex that will need to be further investigated.