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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.

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.

Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam), is a widespread disease that affects cassava (Manihot esculenta Crantz). Studies on the pathogen population structure, pathogen diagnosis, identification and expression of plant genes involved in resistance have been carried out. Different molecular techniques were developed to assess the genetic diversity among the Xampopulations. Characterization of Xam population dynamics over time had enable us to determine the different factors that are associated with resistance breakdown and those that influence the genetic structure or virulence phenotypes of the pathogen's population. Methods for detecting the pathogen in vegetative planting materials and true seeds were developed and contributed to reduce the impact of the disease. To better understand the genetics of resistance a quantitative trait loci (QTLs) approach was developed. Using a PCR-based strategy with degenerate primers we isolated two resistance gene candidates in cassava. We also characterized a region of a chromosome rich in R-gene like sequence. In this review we also report the main results obtained by transcript profiling methodologies, cDNA-AFLP and ESTs developed by the authors to characterize the genes involved in disease resistance. All together these techniques allowed the identification of molecular markers either associated to CBB resistance or that may represent putative genes involved in disease resistance. This article reviews current knowledge on the molecular cassava-Xam interactions.

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)

L'espèce cultivée asiatique, #Oryza sativa#, a une structure génétique tout à fait particulière, objet des travaux décrits dans ce chapitre. Sa domestication, à partir de l'espèce #O. rufipogon#, est très ancienne. Deux sous-espèces ont été identifiées, #indica# et #japonica# à partir des caractères morphologiques et physiologiques et du comportement en croisement. Les marqueurs moléculaires apportent beaucoup à la compréhension de la diversité génétique du riz. Les isoenzymes, malgré le nombre limité de locus et d'allèles accessibles, ont fourni les premiers éléments de quantification de la différenciation intravariétale. La classification établie sur la base des isoenzymes concorde globalement avec des classifications locales, empiriques, ainsi qu'avec les classifications synthètiques, établies sur des critères multiples associant composantes morphologiques et physiologiques. Des études fondées sur plusieurs types de marqueurs moléculaires, ont permis de construire différentes classifications infraspécifiques. Dans ce chapitre, on compare plusieurs schémas de classification reposant sur différents types de caractères, en concentrant les analyses sur des échantillons largement partagés. Ensuite sont examinées quelques situations particulières permettant d'aborder certains aspects de la dynamique qui conditionne la diversité génétique des riz cultivés.

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)

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.

DOI: 10.1139/gen-44-4-589; International audience

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.

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.

Resistance to antifolate antimalarial drugs (cycloguanil, a biologically active metabolite of proguanil, and pyrimethamine) is associated with a Ser- to Asn-108 point mutation in the dihydrofolate reductase thymidylate synthase gene of #Plasmodium falciparum$. The frequency of this mutation was studied in 127 clinical isolates obtained in Yaounde, Cameroon using a simple and rapid molecular technique based on the polymerase chain reaction and restriction fragment lengh polymorphism. Of the 127 isolates, pure wild-type Ser-108 codon, pure mutant-type Asn-108 codon, and mixed codons were observed in 66, 55, and six parasites, respectively. The proportion of antifolate-resistant, pure mutant-type codon, with respect to pure wild-type or mixed alleles, was 43% (55 to 127). The results of the molecular assay were compared with those of semimicro isotopic in vitro assay in 34 isolates. All 17 pure Ser-108 isolates and two isolates with mixed alleles were sensitive to both pyrimethamine (50% inhibitory concentration [IC50] < 100 nM) and cycloguanil (IC50 < 50 nM). Fourteen of 15 isolates with the mutant-type Asn-108 codon were resistant to pyrimethamine and cycloguanil. One isolate with Asn-108 showed a slightly elevated pyrimethamine IC50 (78 nM), which was within the sensitive range. This study provides further evidence that antifolate-resistant #P. falciparum$ isolates are already present in Yaounde, Cameroon. (Résumé d'auteur)

DNA polymorphism and variation in virulence of #Xanthomonas axonopodis$ pv. #manihotis$ (Xam), the causal agent of cassava bacterial blight, were studied within a pathogen population from Venezuela. Collections were made in several fields at different sites within an edaphoclimatic zone where cassava is a major crop. DNA polymorphism was assessed by RFLP analysis, using an Xam plasmidic DNA sequence (ptbB) as a probe to determine the relatedness of 91 Venezuelan isolates. A high degree of polymorphism existed among the isolates, whether collected from the same or different fields. Based on a multiple correspondence analysis, the Xam population was distributed into eight clusters and no correlation was observed between genetic diversity and geographic origin. One set of haplotype strains representing the range of variability detected in Venezuela was further characterized by another RFLP analysis using two repetitive genomic probes (pBS6 and pBS8) to establish the usefulness of these probes and their complementarity with the ptbB probe. Variation for virulence was observed in the Xam Venezuelan collection by inoculating a set of cassava cultivars with 28 isolates of the pathogen, each representig a haplotype. Understanding the genetic and pathogenic variation in the pathogen population is useful for designing cassava bacterial blight management strategies. (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.