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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)

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.

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.

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.

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.

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.

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.

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

National audience

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)

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)

Deux isolats de la granulose de #S. littoralis$ ont été isolés de larves mortes provenant de champs de cotton non traités dans les governorats Gharbeia et Sharkeia en Egypte. Les 2 isolats ont été caractérisés par leur profils de restriction. Les profils obtenus différent de celui publié précédement en provenance de Côte d'Ivoire en Afrique. Les profils polypeptidiques des capisdes ont été déterminé et comparé avec celui de leur homologue africain. Des tests biologiques ont été entrepris. (Résumé d'auteur)

La réaction en chaîne des polymérases/polymorphisme des fragments de restriction (PCR-RFLP) a été utilisée pour séparer des isolats du nématode #Bursaphelenchus$. Les isolats de #B. xylophilus$ examinés provenaient du Japon, des USA, de Chine et du Canada, et ceux de #B. mucronatus$ du Japon, de Chine et de la France. L'ADN ribosomal contenant le gène 5.8S, les segments de transciption interne 1 et 2, et les segments partiels des gènes 18S et 28S ont été amplifiés par PCR. La digestion des produits amplifiés provenant de chaque isolat à l'aide de douze endonucléases de restriction et l'examen des données en RFLP qui en découlent révèlent, par une analyse en grappe, une séparation significative entre #B. xylophilus$ et #B. mucronatus$. Parmi les isolats de #B. xylophilus$ examinés, les isolats pathogènes du Japon, ceux de Chine et des USA étaient tous identiques, tandis que les isloats non pathogènes du Japon étaient légèrement distincts et que ceux du Canada formaient une grappe séparée. Parmi les isolats de #B. mucronatus$, deux isolats provenant du Japon étaient très semblables ; de même un autre isolat du Japon et un isolat de Chine étaient identiques. Les données provenant des séquences d'ADN montrent 98 différences (substitution nucléotidiques ou séparations) dans les 884 paires de bases examinées chez les isolats de #B. xylophilus$ et #B. mucronatus$. Les données provenant des séquences d'ADN chez #Aphelenchus avenae$ et #Aphelenchoides fragariae$ diffèrent non seulement de celles des #Bursaphelenchus$ mais aussi entre elles. Afin de préciser les relations phylogéniques de ces espèces, les données séquentielles du gène 5.8S provenant de l'ADN ribosomal ont été examinées... (D'après résume d'auteur)

Afin d'étudier les possibilités offertes par l'hybridation interspécifique chez #Coffea canephora$, un croisement modèle entre deux espèces phylogénétiquement éloignées est étudié : #C. canephora$ (PSE) x #C. liberica$ var. #dewevrei$ (DEW), proche de #C. canephora$. La quantité d'ADN nucléaire des deux espèces est respectivement de 1,13 pg et de 1,43 pg. Elle peut s'expliquer par des différences de constitution chromosomique principalement au sein de séquences non codantes, essentiellement répétées. La construction d'une banque a été préférée à des méthodes plus sélectives de façon à couvrir le génome en entier. A partir d'une banque d'ADN génomique d'hybride F1 restreint aux site GATC, par l'endonucléase Sau3A, 193 clones contenant des séquences répétées ont été retenus, après rétrohybridation. Vingt deux sondes sont totalement séquencées, 5 partiellement. Elles sont riches en AT et montrent souvent des sites GATC internes. La plupart ne présente pas d'homologie avec les séquences connues. Trois sondes sont analysées de façon plus approfondie sur les espèces parentes PSE et DEW et sur 8 autres espèces choisies pour leur origine géographique et leur quantité d'ADN (#C. racemosa$, #C. sessiliflora$, #C. eugenioides$, #C. sp$ "#Moloundou$", #C. canephora$, #C. congensis$ et #C. heterocalyx$). La première est constituée de quatre séquences distinctes, dont une au moins répétée en tandem. La seconde révèle une forte homologie avec des séquences codant pour la reverse-transcriptase de rétrotransposon de type gypsy-like, tant au niveau nucléotidique qu'au niveau acides aminés. Elle est donc, le premier rétrotransposon découvert chez les caféiers. La dernière possède des profils RFLP sur l'ADN différents uniquement pour les deux espèces à forte quantité d'ADN. Il est primordial de poursuivre l'étude du rétrotransposon pour préciser son nombre de répétitions, sa structure fine et sa capacité à transposer... (D'après résumé d'auteur)

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)