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Understanding the evolutionary patterns of genetic diversity of cultivated plants and their wild relatives is a fundamental aspect of agrobiodiversity research. Documenting genetic diversity and understanding the evolutionary histories of crops will have important impacts in terms of food security in southern countries such as Africa. In this study, we develop a generalized method for sequencing full chloroplast genomes via hybridization enrichment suitable for multiplexing a large number of individuals. We will present application of the method to the study of wild pearl millet, wild and cultivated yams, fonio and to African rice. (Texte intégral)

Madagascar Island was one of the last major Old World areas where human settlement was accompanied by the introduction of Oryza sativa. Early studies had reported the presence of a rice group specific to Madagascar. Using 1536 SNP markers, we compared diversity patterns between a panel of 147 Malagasy rice varieties, a reference panel of 370 Asian varieties and representatives of wild relatives of O. sativa. Migration bottleneck has resulted in 30-40% reduction of diversity among the indica and japonica groups in Madagascar. The Malagasy panel showed many fewer indica x japonica recombinations compared to the Asian panel, suggesting that the two groups had undergone much less recombinations when migration to the Island occurred. The existence of the Malagasy-specific group (Gm) was confirmed. Its diversity patterns positioned it halfway from indica and aus groups. Madagascar also hosted cold tolerant tropical japonica varieties, with very long grain. The Gm group most probably arose from founder effect from intermediary forms of rice originated from either India or Sri Lanka that did not belong to the four majors O. sativa groups. It then underwent human selection for cold tolerance. Signs of inter-group recombinations were also observed, but recombinations did not seem to have played a major role in the dynamics of rice adaptation to the Island's agro-ecological constraints. Connections between Gm and O. rufipogon from its putative area of origin reinforce the hypothesis of multiple and diffuse domestication of O. sativa as opposed to two independent domestications occurring in two distinct geographical areas. (Texte intégral)

Madagascar Island was one of the last major Old World areas where human settlement was accompanied by the introduction of Oryza sativa. Early studies had reported the presence of a rice group specific to Madagascar. Using 1536 SNP markers, we compared diversity patterns between a panel of 147 Malagasy rice varieties, a reference panel of 370 Asian varieties and representatives of wild relatives of O. sativa. Migration bottleneck has resulted in 30-40% reduction of diversity among the indica and japonica groups in Madagascar. The Malagasy panel showed many fewer indica x japonica recombinations compared to the Asian panel, suggesting that the two groups had undergone much less recombinations when migration to the Island occurred. The existence of the Malagasy-specific group (Gm) was confirmed. Its diversity patterns positioned it halfway from indica and aus groups. Madagascar also hosted cold tolerant tropical japonica varieties, with very long grain. The Gm group most probably arose from founder effect from intermediary forms of rice originated from either India or Sri Lanka that did not belong to the four majors O. sativa groups. It then underwent human selection for cold tolerance. Signs of inter-group recombinations were also observed, but recombinations did not seem to have played a major role in the dynamics of rice adaptation to the Island's agro-ecological constraints. Connections between Gm and O. rufipogon from its putative area of origin reinforce the hypothesis of multiple and diffuse domestication of O. sativa as opposed to two independent domestications occurring in two distinct geographical areas. (Texte intégral)

A haploid C. clementina was chosen by the International Citrus Genomic Consortium (ICGC) to establish the reference whole genome sequence for Citrus. Development of a dense clementine linkage map was part of the objectives of this global collaborative project. Two inter-specific populations between C. clementina and C. maxima were used for this purpose. 156 hybrids of Nules clementine x Pink pummelo and 200 hybrids of Chandler pummelo x Nules clementine were genotyped with 1003 markers. 306 were SSRs markers (66 from genomic libraries, 207 from ESTs and 33 from clementine BAC end sequences -BES-), 34 were Indels markers mined from BES and 663 SNPs mined from Clementine BES or identified by candidate gene sequencing. 901 markers were successfully mapped in the 9 clementine linkage groups. Segregation distortion was observed for many loci of clementine when it was used as male parent while it followed Mendelian segregation for most markers when used as female parent. However marker order was mostly conserved between the male and female maps; thus, data of the two populations were joined to establish the reference clementine genetic map. The total clementine linkage map cover 1250 cM with linkage groups from 105 cM until 210 cM. This map is strongly anchored on a large diploid clementine BAC library resource. It is a powerful tool for Citrus genetics and supports the alignment of the haploid clementine whole genome sequence in the framework of the ICGC collaborative project.

The core approach for investigating genetic diversity in a crop germplasm collection has proven merits, among which the possibility to choose a sample of manageable size (e.g. a 'minicore'), so that it can be studied in details, be exchanged among researchers and users and serve as a common reference for integration of data of multiple sources. In turn, studies on a mini core collection can help direct further investigation in the whole collection and target specific compartments for specific purposes. The Generation Challenge Programme is helping various germplasm centers develop genetic stocks to serve as core reference sets for an array of important food crops ; the case of sorghum will be presented as an advanced example. Sorghum reference set has been evaluated under normal and postflowering water stress conditions at ICRISAT. A wide range of variation for agronomic traits, including yield and Fe and Zn have been observed and promising lines selected for use in breeding programmes. The forthcoming possibility to apply massive genotyping to crop germplasm may justify adaptation of scientific strategies, in relation to the biology and the history of the crops. These aspects will be discussed for two contrasting crops, namely sorghum, a diploid, predominantly inbreeding cereal crop with vast germplasm collections, and banana/plantain, a multi-, essentially tri-ploid, vegetatively propagated fruit crop with collections of limited size. (Texte intégral)

A haploid C. clementina was chosen by the International Citrus Genomic Consortium (ICGC) to establish the reference whole genome sequence for Citrus. Development of a dense clementine linkage map was part of the objectives of this global collaborative project. Two inter-specific populations between C. clementina and C. maxima were used for this purpose. 156 hybrids of Nules clementine x Pink pummelo and 200 hybrids of Chandler pummelo x Nules clementine were genotyped with 1003 markers. 306 were SSRs markers (66 from genomic libraries, 207 from ESTs and 33 from clementine BAC end sequences -BES-), 34 were Indels markers mined from BES and 663 SNPs mined from Clementine BES or identified by candidate gene sequencing. 901 markers were successfully mapped in the 9 clementine linkage groups. Segregation distortion was observed for many loci of clementine when it was used as male parent while it followed Mendelian segregation for most markers when used as female parent. However marker order was mostly conserved between the male and female maps; thus, data of the two populations were joined to establish the reference clementine genetic map. The total clementine linkage map cover 1250 cM with linkage groups from 105 cM until 210 cM. This map is strongly anchored on a large diploid clementine BAC library resource. It is a powerful tool for Citrus genetics and supports the alignment of the haploid clementine whole genome sequence in the framework of the ICGC collaborative project.

The ADOC project (GCP 2006-02) aimed to characterize allelic diversity at orthologous loci of candidate genes for drought tolerance in seven GCP crops (rice, barley, sorghum, bean, chickpea, cassava and potato), working on reference collections of around 300 accessions for each crop. Six gene families (ERECTA, DREB, SS, SPS, ASR and VIN) were selected as the initial subset of target genes. Except the DREB gene family, for which a specific focus has been given to DREB2A, and the SPS gene family in cereals, for which only the Os01g69030 orthology group was studied, they represent a set of relatively small gene families acting at different levels of the drought stress response (transcriptional regulation, carbohydrate metabolism...) for which a comparative analysis of gene families was undertaken. Obtaining complete gene families was easier in whole sequenced genomes like rice and sorghum. Polyploidy and heterozygosity induced difficulties in analyzing data for cassava and potato; However sequences for a few genes were obtained and analyzed for SNP diversity across all species. Population structure influenced partially haplotype patterns. A large range of haplotype diversity was found and the degrees of this differed between species. Different patterns and range of sequence diversity were found within gene families and between species for orthologous genes. For a few genes, computation of a sequence-based neutrality test suggested selection events acting at the species and/or subgroup level. (Texte intégral)

Sorghum is, together with pearl millet, one of the most important cereals in West Africa. It is the second most important crop in Africa after maize. Sorghum production in West Africa is principally based on traditional, low harvest index cultivars and breeding efforts of the past 40 years have had limited positive impact. At the same time, sorghum with an aligned genome sequence available since 2007, constitutes a model for grass species and is rich of huge resources in terms of genomic tools and information. Building on a five-year effort to characterize worldwide sorghum diversity and develop resources for association mapping studies, several approaches are now explored for integrating molecular tools and approaches into sorghum breeding programs in Mali. This presentation will focus on two approaches developed as part of the GCP challenge initiative "Drought Tolerance Improvement for Sorghum in Africa". The first strategy integrates recent sorghum breeding achievements in Mali and methodologies for marker-assisted recurrent selection (MARS) that provide significant improvement of breeding efficiency for complex traits, as demonstrated in maize. The second, develops a modified nested association mapping (NAM) design exploiting backcross products from a set of elite recurrent parents in combination with several genetically diverse donor parents chosen as sources of cryptic alleles for improvement of productivity and adaptation to prevalent biotic and abiotic stresses. This project will combine an applied component toward the development of new broadened basis varieties with the development of experimental populations with strong genetic resolution and long-term value for identifying marker-trait associations. (Texte intégral)

Sorghum is, together with pearl millet, one of the most important cereals in West Africa. It is the second most important crop in Africa after maize. Sorghum production in West Africa is principally based on traditional, low harvest index cultivars and breeding efforts of the past 40 years have had limited positive impact. At the same time, sorghum with an aligned genome sequence available since 2007, constitutes a model for grass species and is rich of huge resources in terms of genomic tools and information. Building on a five-year effort to characterize worldwide sorghum diversity and develop resources for association mapping studies, several approaches are now explored for integrating molecular tools and approaches into sorghum breeding programs in Mali. This presentation will focus on two approaches developed as part of the GCP challenge initiative "Drought Tolerance Improvement for Sorghum in Africa". The first strategy integrates recent sorghum breeding achievements in Mali and methodologies for marker-assisted recurrent selection (MARS) that provide significant improvement of breeding efficiency for complex traits, as demonstrated in maize. The second, develops a modified nested association mapping (NAM) design exploiting backcross products from a set of elite recurrent parents in combination with several genetically diverse donor parents chosen as sources of cryptic alleles for improvement of productivity and adaptation to prevalent biotic and abiotic stresses. This project will combine an applied component toward the development of new broadened basis varieties with the development of experimental populations with strong genetic resolution and long-term value for identifying marker-trait associations. (Texte intégral)

The ADOC project (GCP 2006-02) aimed to characterize allelic diversity at orthologous loci of candidate genes for drought tolerance in seven GCP crops (rice, barley, sorghum, bean, chickpea, cassava and potato), working on reference collections of around 300 accessions for each crop. Six gene families (ERECTA, DREB, SS, SPS, ASR and VIN) were selected as the initial subset of target genes. Except the DREB gene family, for which a specific focus has been given to DREB2A, and the SPS gene family in cereals, for which only the Os01g69030 orthology group was studied, they represent a set of relatively small gene families acting at different levels of the drought stress response (transcriptional regulation, carbohydrate metabolism...) for which a comparative analysis of gene families was undertaken. Obtaining complete gene families was easier in whole sequenced genomes like rice and sorghum. Polyploidy and heterozygosity induced difficulties in analyzing data for cassava and potato; However sequences for a few genes were obtained and analyzed for SNP diversity across all species. Population structure influenced partially haplotype patterns. A large range of haplotype diversity was found and the degrees of this differed between species. Different patterns and range of sequence diversity were found within gene families and between species for orthologous genes. For a few genes, computation of a sequence-based neutrality test suggested selection events acting at the species and/or subgroup level. (Texte intégral)

We report here a study on sorghum, using a well characterized sample of diverse cultivars that we resequenced for portions of six genes possibly involved in grain quality variation. Our study was based on 1.7 Mbp sequence data enabling a comparison between 129 to 184 accessions of sorghum for six genes and 1008 to 3771 bp per gene. A total of 170 polymorphisms, including 141 SNPs and 29 Insertion/Deletion Polymorphisms, were recorded within a total of 11279 bp scored. We analysed the distribution of polymorphisms by comparing the area of origin of sorghum and areas where sorghum migrated in the course of domestication and subsequent human migrations. We observed an array of situations. On one hand we have polymorphisms which are distributed in agreement with a neutral hypothesis, on the other extreme we have Opaque2, for which new polymorphisms have appeared in areas outside the area of origin. This, we argue, is novel diversity which was selected by man in the course of domestication: crop neo-diversity. Documentation of the extent of crop neo-diversity is very important. In breeding it might explain how narrow-based populations can be improved in the long term; in germplasm management it implies reformatting concepts and approaches, whereby diversity is not just reduced from wild to landraces and to improved materials; it may stimulate studies which investigate patterns of diversity in regions where crops were 'adapted' by man, in the search for adaptive neo-diversity. (Texte intégral)

We report here a study on sorghum, using a well characterized sample of diverse cultivars that we resequenced for portions of six genes possibly involved in grain quality variation. Our study was based on 1.7 Mbp sequence data enabling a comparison between 129 to 184 accessions of sorghum for six genes and 1008 to 3771 bp per gene. A total of 170 polymorphisms, including 141 SNPs and 29 Insertion/Deletion Polymorphisms, were recorded within a total of 11279 bp scored. We analysed the distribution of polymorphisms by comparing the area of origin of sorghum and areas where sorghum migrated in the course of domestication and subsequent human migrations. We observed an array of situations. On one hand we have polymorphisms which are distributed in agreement with a neutral hypothesis, on the other extreme we have Opaque2, for which new polymorphisms have appeared in areas outside the area of origin. This, we argue, is novel diversity which was selected by man in the course of domestication: crop neo-diversity. Documentation of the extent of crop neo-diversity is very important. In breeding it might explain how narrow-based populations can be improved in the long term; in germplasm management it implies reformatting concepts and approaches, whereby diversity is not just reduced from wild to landraces and to improved materials; it may stimulate studies which investigate patterns of diversity in regions where crops were 'adapted' by man, in the search for adaptive neo-diversity. (Texte intégral)

The Generation Challenge Programme's Global Composite Germplasm Collection of 3372 wild and cultivated sorghums includes 280 elite breeding lines and improved cultivars, 250 Chinese germplasm lines to be provided by CAAS, 64 wild accessions, and >3000 landrace accessions selected from previously defined core collections, for resistance/tolerance to production constraints, and/or for variation in other traits. A set of 48 sorghum SSR primer pairs detecting loci distributed across all ten linkage groups was chosen following preliminary analysis of 48 diverse sorghum landrace accessions with 104 available SSRs complemented by additional SSRs from CIRAD and ICRISAT. Diversity analysis was performed on 3367 accessions genotyped with 41 SSR primer pairs by CIRAD and ICRISAT. Breeding lines and wild accessions clustered separately from landraces, which exhibited structure explainable by geographic origin. Landrace population substructure was further characterized within racial groups (five basic races and ten hybrid races). Race bicolor showed little evidence of population structure, congruent with it being the original domesticate. Race kafir (largely from Southern Africa) was distinct. Accessions of the durra, caudatum and guinea races each formed four distinct geographic subgroups. The guinea race margaritiferum group formed its own cluster, which clustered with most of the wild accessions, suggesting its independent domestication. Intermediate races behaved similarly. A reference subset of 384 accessions was then defined for allele mining and more detailed characterization. Limited quantities of seed and DNA of this 384-entry sorghum reference germplasm set will be available for distribution from ICRISAT under the terms and conditions of the Standard Material Transfer Agreement of the International Treaty on Crop Genetic Resources. (Texte intégral)

Drought is a major abiotic stress. Many candidate genes have been proposed during the last years to explain aspects of tolerance to drought stress, for a specific crop and in a specific environment. However, the relation between gene structural polymorphism and functional diversity is seldom clear. In a program founded by GCP( Generation Challenge Program) we are currently producing a dataset of allelic diversity at orthologous candidate genes for drought tolerance in rice, sorghum and barley. The ASR (ABA, Stress and Ripening) gene family is one of the target genes chosen in this program. Many evidences show the implication of this family in drought tolerance, notably the co-localization between ASR1 locus and a QTL directly linked to drought tolerance in maize and the increase in ASR transcript level under osmotic stress in rice. We gathered all sequences available in the databases on this gene family. We used these sequences to design consensus primers in order to amplify all genes of this family in the three cereals. Based on phylogenetic relationships between genes, orthologous genes are identified. In a second step, their whole sequence polymorphism is assessed in a DNA bank of 283 reference accessions for each crop already characterized with neutral markers. The first analysis of allelic diversity shows that some rice ASR genes are on purifying selection and other may be on relaxed selection. This reference germplasm could allow testing association between genes polymorphism and trait variability. (Texte intégral)

Following the difficulties encountered for assembling a 1.2 x sequencing of the highly heterozygous sweet orange genome, the International Citrus Genomic Consortium (ICOC) decided to estab1ish the first reference sequence of a whole nuclear citrus genome from a haploid Clementine. A saturated genetic linkage map of Clementine based on sequence-characterized markers was considered by the ICGC as an important too1 for genome sequence assemb1y. In this framework, CIRAD proposed to use an interspecific population C. maxima x C. clementina to implement the reference Clementine genetic map. A population of 250 hybrids of Chandler pummelo x Clementine was established in Corsica and 190 hybrids were used in this first phase of mapping. Collaboration was established between two French organizations (CIRAD and INRA), two groups from United States (UF-CREC and UCR), one Spanish institute (IVIA), INRA Morocco and Cukurova University from Turkey. Forty markers were found heterozygous in Clementine among a previous set of 90 SSR markers developed by CIRAD from microsatellite-enriched genomic libraries. With the objective to integrate the physical and genetic maps of Clementine, CIRAD and IVIA have developed new SSR markers from microsatellite sequences identified in BAC End Sequences (BES) of diploid Clementine. On hundred and 10 of these new markers were found heterozygous for Clementine or Chandler pummelo and were used for genotyping. INRA France deve1oped 500 SSR markers from ESTs databases and found 170 markers heterozygous for Clementine. INRA Morocco contributed to the genotyping of 112 SSR markers developed from EST databases and genomic libraries, while 50 ESTs SSR were analysed by Cukurova University. SSR markers mainly developed from EST databases and already mapped for sweet orange were genotyped by UF-CREC (70 markers) and UCR (60 markers) to allow comparisons among the C. sinensis. C. maxima and C. c1ementina maps. lndeed, taking advantage of the important allelic d

Following the difficulties encountered for assembling a 1.2 x sequencing of the highly heterozygous sweet orange genome, the International Citrus Genomic Consortium (ICOC) decided to estab1ish the first reference sequence of a whole nuclear citrus genome from a haploid Clementine. A saturated genetic linkage map of Clementine based on sequence-characterized markers was considered by the ICGC as an important too1 for genome sequence assemb1y. In this framework, CIRAD proposed to use an interspecific population C. maxima x C. clementina to implement the reference Clementine genetic map. A population of 250 hybrids of Chandler pummelo x Clementine was established in Corsica and 190 hybrids were used in this first phase of mapping. Collaboration was established between two French organizations (CIRAD and INRA), two groups from United States (UF-CREC and UCR), one Spanish institute (IVIA), INRA Morocco and Cukurova University from Turkey. Forty markers were found heterozygous in Clementine among a previous set of 90 SSR markers developed by CIRAD from microsatellite-enriched genomic libraries. With the objective to integrate the physical and genetic maps of Clementine, CIRAD and IVIA have developed new SSR markers from microsatellite sequences identified in BAC End Sequences (BES) of diploid Clementine. On hundred and 10 of these new markers were found heterozygous for Clementine or Chandler pummelo and were used for genotyping. INRA France deve1oped 500 SSR markers from ESTs databases and found 170 markers heterozygous for Clementine. INRA Morocco contributed to the genotyping of 112 SSR markers developed from EST databases and genomic libraries, while 50 ESTs SSR were analysed by Cukurova University. SSR markers mainly developed from EST databases and already mapped for sweet orange were genotyped by UF-CREC (70 markers) and UCR (60 markers) to allow comparisons among the C. sinensis. C. maxima and C. c1ementina maps. lndeed, taking advantage of the important allelic d

Drought is a major abiotic stress. Many candidate genes have been proposed during the last years to explain aspects of tolerance to drought stress, for a specific crop and in a specific environment. However, the relation between gene structural polymorphism and functional diversity is seldom clear. In a program founded by GCP( Generation Challenge Program) we are currently producing a dataset of allelic diversity at orthologous candidate genes for drought tolerance in rice, sorghum and barley. The ASR (ABA, Stress and Ripening) gene family is one of the target genes chosen in this program. Many evidences show the implication of this family in drought tolerance, notably the co-localization between ASR1 locus and a QTL directly linked to drought tolerance in maize and the increase in ASR transcript level under osmotic stress in rice. We gathered all sequences available in the databases on this gene family. We used these sequences to design consensus primers in order to amplify all genes of this family in the three cereals. Based on phylogenetic relationships between genes, orthologous genes are identified. In a second step, their whole sequence polymorphism is assessed in a DNA bank of 283 reference accessions for each crop already characterized with neutral markers. The first analysis of allelic diversity shows that some rice ASR genes are on purifying selection and other may be on relaxed selection. This reference germplasm could allow testing association between genes polymorphism and trait variability. (Texte intégral)

The Generation Challenge Programme's Global Composite Germplasm Collection of 3372 wild and cultivated sorghums includes 280 elite breeding lines and improved cultivars, 250 Chinese germplasm lines to be provided by CAAS, 64 wild accessions, and >3000 landrace accessions selected from previously defined core collections, for resistance/tolerance to production constraints, and/or for variation in other traits. A set of 48 sorghum SSR primer pairs detecting loci distributed across all ten linkage groups was chosen following preliminary analysis of 48 diverse sorghum landrace accessions with 104 available SSRs complemented by additional SSRs from CIRAD and ICRISAT. Diversity analysis was performed on 3367 accessions genotyped with 41 SSR primer pairs by CIRAD and ICRISAT. Breeding lines and wild accessions clustered separately from landraces, which exhibited structure explainable by geographic origin. Landrace population substructure was further characterized within racial groups (five basic races and ten hybrid races). Race bicolor showed little evidence of population structure, congruent with it being the original domesticate. Race kafir (largely from Southern Africa) was distinct. Accessions of the durra, caudatum and guinea races each formed four distinct geographic subgroups. The guinea race margaritiferum group formed its own cluster, which clustered with most of the wild accessions, suggesting its independent domestication. Intermediate races behaved similarly. A reference subset of 384 accessions was then defined for allele mining and more detailed characterization. Limited quantities of seed and DNA of this 384-entry sorghum reference germplasm set will be available for distribution from ICRISAT under the terms and conditions of the Standard Material Transfer Agreement of the International Treaty on Crop Genetic Resources. (Texte intégral)