Development of nuclear microsatellite markers for the fonio, Digitaria exilis (Poaceae), an understudied West African cereal.

PREMISE OF THE STUDY: We developed nuclear microsatellite primers to explore the genetic diversity, population genetic structure, and evolutionary history of the fonio (Digitaria exilis), an understudied cereal cultivated in West Africa. METHODS AND RESULTS: We used a microsatellite-enriched library approach to isolate and characterize 38 nuclear primer pairs (31 di-, five tri-, and two tetranucleotide repeats), of which 21 were polymorphic and exhibited a clear pattern in 36 accessions from West Africa. The number of alleles per locus ranged from two to 22, with a mean of 4.71, and expected heterozygosity ranged from 0.03 to 0.93. CONCLUSIONS: The developed set of 21 polymorphic SSR markers will provide tools for population and evolutionary genetics studies of the cultivated fonio.

Genomic footprints of selection in early-and late-flowering pearl millet landraces.

Pearl millet is among the top three-cereal production in one of the most climate vulnerable regions, sub-Saharan Africa. Its Sahelian origin makes it adapted to grow in poor sandy soils under low soil water regimes. Pearl millet is thus considered today as one of the most interesting crops to face the global warming. Flowering time, a trait highly correlated with latitude, is one of the key traits that could be modulated to face future global changes. West African pearl millet landraces, can be grouped into early- (EF) and late-flowering (LF) varieties, each flowering group playing a specific role in the functioning and resilience of Sahelian smallholders. The aim of this study was thus to detect genes linked to flowering but also linked to relevant traits within each flowering group. We thus investigated genomic and phenotypic diversity in 109 pearl millet landrace accessions, i.e., 66 early-flowering and 43 late-flowering, grown in the groundnut basin, the first area of rainfed agriculture in Senegal dominated by dry cereals (millet, maize, and sorghum) and legumes (groundnuts, cowpeas). We were able to confirm the role of PhyC gene in pearl millet flowering and identify several other genes that appear to be as much as important, such as FSR12 and HAC1. HAC1 and two other genes appear to be part of QTLs previously identified and deserve further investigation. At the same time, we were able to highlight a several genes and variants that could contribute to the improvement of pearl millet yield, especially since their impact was demonstrated across flowering cycles.

Abandonment of pearl millet cropping and homogenization of its diversity over a 40 year period in Senegal.

Cultivated diversity is considered an insurance against major climatic variability. However, since the 1980s, several studies have shown that climate variability and agricultural changes may already have locally eroded crop genetic diversity. We studied pearl millet diversity in Senegal through a comparison of pearl millet landraces collected 40 years apart. We found that more than 20% of villages visited in 1976 had stopped growing pearl millet. Despite this, its overall genetic diversity has been maintained but differentiation between early- and late-flowering accessions has been reduced. We also found stronger crop-to-wild gene flow than wild-to-crop gene flow and that wild-to-crop gene flow was weaker in 2016 than in 1976. In conclusion, our results highlight genetic homogenization in Senegal. This homogenization within cultivated pearl millet and between wild and cultivated forms is a key factor in genetic erosion and it is often overlooked. Improved assessment and conservation strategies are needed to promote and conserve both wild and cultivated pearl millet diversity.

Pearl millet genomic vulnerability to climate change in West Africa highlights the need for regional collaboration.

Climate change is already affecting agro-ecosystems and threatening food security by reducing crop productivity and increasing harvest uncertainty. Mobilizing crop diversity could be an efficient way to mitigate its impact. We test this hypothesis in pearl millet, a nutritious staple cereal cultivated in arid and low-fertility soils in sub-Saharan Africa. We analyze the genomic diversity of 173 landraces collected in West Africa together with an extensive climate dataset composed of metrics of agronomic importance. Mapping the pearl millet genomic vulnerability at the 2050 horizon based on the current genomic-climate relationships, we identify the northern edge of the current areas of cultivation of both early and late flowering varieties as being the most vulnerable to climate change. We predict that the most vulnerable areas will benefit from using landraces that already grow in equivalent climate conditions today. However, such seed-exchange scenarios will require long distance and trans-frontier assisted migrations. Leveraging genetic diversity as a climate mitigation strategy in West Africa will thus require regional collaboration.

Fonio millet genome unlocks African orphan crop diversity for agriculture in a changing climate.

Sustainable food production in the context of climate change necessitates diversification of agriculture and a more efficient utilization of plant genetic resources. Fonio millet (Digitaria exilis) is an orphan African cereal crop with a great potential for dryland agriculture. Here, we establish high-quality genomic resources to facilitate fonio improvement through molecular breeding. These include a chromosome-scale reference assembly and deep re-sequencing of 183 cultivated and wild Digitaria accessions, enabling insights into genetic diversity, population structure, and domestication. Fonio diversity is shaped by climatic, geographic, and ethnolinguistic factors. Two genes associated with seed size and shattering showed signatures of selection. Most known domestication genes from other cereal models however have not experienced strong selection in fonio, providing direct targets to rapidly improve this crop for agriculture in hot and dry environments.

Adaptive Introgression: An Untapped Evolutionary Mechanism for Crop Adaptation.

Global environmental changes strongly impact wild and domesticated species biology and their associated ecosystem services. For crops, global warming has led to significant changes in terms of phenology and/or yield. To respond to the agricultural challenges of this century, there is a strong need for harnessing the genetic variability of crops and adapting them to new conditions. Gene flow, from either the same species or a different species, may be an immediate primary source to widen genetic diversity and adaptions to various environments. When the incorporation of a foreign variant leads to an increase of the fitness of the recipient pool, it is referred to as "adaptive introgression". Crop species are excellent case studies of this phenomenon since their genetic variability has been considerably reduced over space and time but most of them continue exchanging genetic material with their wild relatives. In this paper, we review studies of adaptive introgression, presenting methodological approaches and challenges to detecting it. We pay particular attention to the potential of this evolutionary mechanism for the adaptation of crops. Furthermore, we discuss the importance of farmers' knowledge and practices in shaping wild-to-crop gene flow. Finally, we argue that screening the wild introgression already existing in the cultivated gene pool may be an effective strategy for uncovering wild diversity relevant for crop adaptation to current environmental changes and for informing new breeding directions.

A western Sahara centre of domestication inferred from pearl millet genomes.

There have been intense debates over the geographic origin of African crops and agriculture. Here, we used whole-genome sequencing data to infer the domestication origin of pearl millet (Cenchrus americanus). Our results supported an origin in western Sahara, and we dated the onset of cultivated pearl millet expansion in Africa to 4,900 years ago. We provided evidence that wild-to-crop gene flow increased cultivated genetic diversity leading to diversity hotspots in western and eastern Sahel and adaptive introgression of 15 genomic regions. Our study reconciled genetic and archaeological data for one of the oldest African crops.

Structure of sweet potato (Ipomoea batatas) diversity in West Africa covaries with a climatic gradient.

Sub-Saharan agriculture has been identified as vulnerable to ongoing climate change. Adaptation of agriculture has been suggested as a way to maintain productivity. Better knowledge of intra-specific diversity of varieties is prerequisites for the successful management of such adaptation. Among crops, root and tubers play important roles in food security and economic growth for the most vulnerable populations in Africa. Here, we focus on the sweet potato. The Sweet potato (Ipomoea batatas) was domesticated in Central and South America and was later introduced into Africa and is now cultivated throughout tropical Africa. We evaluated its diversity in West Africa by sampling a region extending from the coastal area of Togo to the northern Sahelian region of Senegal that represents a range of climatic conditions. Using 12 microsatellite markers, we evaluated 132 varieties along this gradient. Phenotypic data from field trials conducted in three seasons was also obtained. Genetic diversity in West Africa was found to be 18% lower than in America. Genetic diversity in West Africa is structured into five groups, with some groups found in very specific climatic areas, e.g. under a tropical humid climate, or under a Sahelian climate. We also observed genetic groups that occur in a wider range of climates. The genetic groups were also associated with morphological differentiation, mainly the shape of the leaves and the color of the stem or root. This particular structure of diversity along a climatic gradient with association to phenotypic variability can be used for conservation strategies. If such structure is proved to be associated with specific climatic adaptation, it will also allow developing strategies to adapt agriculture to ongoing climate variation in West Africa.

New Genetic Insights into Pearl Millet Diversity As Revealed by Characterization of Early- and Late-Flowering Landraces from Senegal.

Pearl millet (Pennisetum glaucum (L.) R. Br.) is a staple food and a drought-tolerant cereal well adapted to Sub-Saharan Africa agro-ecosystems. An important diversity of pearl millet landraces has been widely conserved by farmers and therefore could help copping with climate changes and contribute to future food security. Hence, characterizing its genetic diversity and population structure can contribute to better assist breeding programs for a sustainable agricultural productivity enhancement. Toward this goal, a comprehensive panel of 404 accessions were used that correspond to 12 improved varieties, 306 early flowering and 86 late-flowering cultivated landraces from Senegal. Twelve highly polymorphic SSR markers were used to study diversity and population structure. Two genes, PgMADS11 and PgPHYC, were genotyped to assess their association to flowering phenotypic difference in landraces. Results indicate a large diversity and untapped potential of Senegalese pearl millet germplasm as well as a genetic differentiation between early- and late-flowering landraces. Further, a fine-scale genetic difference of PgPHYC and PgMADS11 (SNP and indel, respectively) and co-variation of their alleles with flowering time were found among landraces. These findings highlight new genetic insights of pearl millet useful to define heterotic populations for breeding, genomic association panel, or crosses for trait-specific mapping.

A large set of 26 new reference transcriptomes dedicated to comparative population genomics in crops and wild relatives.

We produced a unique large data set of reference transcriptomes to obtain new knowledge about the evolution of plant genomes and crop domestication. For this purpose, we validated a RNA-Seq data assembly protocol to perform comparative population genomics. For the validation, we assessed and compared the quality of de novo Illumina short-read assemblies using data from two crops for which an annotated reference genome was available, namely grapevine and sorghum. We used the same protocol for the release of 26 new transcriptomes of crop plants and wild relatives, including still understudied crops such as yam, pearl millet and fonio. The species list has a wide taxonomic representation with the inclusion of 15 monocots and 11 eudicots. All contigs were annotated using BLAST, prot4EST and Blast2GO. A strong originality of the data set is that each crop is associated with close relative species, which will permit whole-genome comparative evolutionary studies between crops and their wild-related species. This large resource will thus serve research communities working on both crops and model organisms. All the data are available at http://arcad-bioinformatics.southgreen.fr/.

Influence of ethnolinguistic diversity on the sorghum genetic patterns in subsistence farming systems in eastern Kenya.

Understanding the effects of actions undertaken by human societies on crop evolution processes is a major challenge for the conservation of genetic resources. This study investigated the mechanisms whereby social boundaries associated with patterns of ethnolinguistic diversity have influenced the on-farm distribution of sorghum diversity. Social boundaries limit the diffusion of planting material, practices and knowledge, thus shaping crop diversity in situ. To assess the effect of social boundaries, this study was conducted in the contact zone between the Chuka, Mbeere and Tharaka ethnolinguistic groups in eastern Kenya. Sorghum varieties were inventoried and samples collected in 130 households. In all, 297 individual plants derived from seeds collected under sixteen variety names were characterized using a set of 18 SSR molecular markers and 15 morphological descriptors. The genetic structure was investigated using both a Bayesian assignment method and distance-based clustering. Principal Coordinates Analysis was used to describe the structure of the morphological diversity of the panicles. The distribution of the varieties and the main genetic clusters across ethnolinguistic groups was described using a non-parametric MANOVA and pairwise Fisher tests. The spatial distribution of landrace names and the overall genetic spatial patterns were significantly correlated with ethnolinguistic partition. However, the genetic structure inferred from molecular makers did not discriminate the short-cycle landraces despite their morphological distinctness. The cases of two improved varieties highlighted possible fates of improved materials. The most recent one was often given the name of local landraces. The second one, that was introduced a dozen years ago, displays traces of admixture with local landraces with differential intensity among ethnic groups. The patterns of congruence or discordance between the nomenclature of farmers' varieties and the structure of both genetic and morphological diversity highlight the effects of the social organization of communities on the diffusion of seed, practices, and variety nomenclature.

Cost-effective enrichment hybridization capture of chloroplast genomes at deep multiplexing levels for population genetics and phylogeography studies.

Biodiversity, phylogeography and population genetic studies will be revolutionized by access to large data sets thanks to next-generation sequencing methods. In this study, we develop an easy and cost-effective protocol for in-solution enrichment hybridization capture of complete chloroplast genomes applicable at deep-multiplexed levels. The protocol uses cheap in-house species-specific probes developed via long-range PCR of the entire chloroplast. Barcoded libraries are constructed, and in-solution enrichment of the chloroplasts is carried out using the probes. This protocol was tested and validated on six economically important West African crop species, namely African rice, pearl millet, three African yam species and fonio. For pearl millet, we also demonstrate the effectiveness of this protocol to retrieve 95% of the sequence of the whole chloroplast on 95 multiplexed individuals in a single MiSeq run at a success rate of 95%. This new protocol allows whole chloroplast genomes to be retrieved at a modest cost and will allow unprecedented resolution for closely related species in phylogeography studies using plastomes.

Population genetics of Ceratitis capitata in South Africa: implications for dispersal and pest management.

The invasive Mediterranean fruit fly (medfly), Ceratitis capitata, is one of the major agricultural and economical pests globally. Understanding invasion risk and mitigation of medfly in agricultural landscapes requires knowledge of its population structure and dispersal patterns. Here, estimates of dispersal ability are provided in medfly from South Africa at three spatial scales using molecular approaches. Individuals were genotyped at 11 polymorphic microsatellite loci and a subset of individuals were also sequenced for the mitochondrial cytochrome oxidase subunit I gene. Our results show that South African medfly populations are generally characterized by high levels of genetic diversity and limited population differentiation at all spatial scales. This suggests high levels of gene flow among sampling locations. However, natural dispersal in C. capitata has been shown to rarely exceed 10 km. Therefore, documented levels of high gene flow in the present study, even between distant populations (>1600 km), are likely the result of human-mediated dispersal or at least some form of long-distance jump dispersal. These findings may have broad applicability to other global fruit production areas and have significant implications for ongoing pest management practices, such as the sterile insect technique.

Biodiversity, evolution and adaptation of cultivated crops.

The human diet depends on very few crops. Current diversity in these crops is the result of a long interaction between farmers and cultivated plants, and their environment. Man largely shaped crop biodiversity from the domestication period 12,000 B.P. to the development of improved varieties during the last century. We illustrate this process through a detailed analysis of the domestication and early diffusion of maize. In smallholder agricultural systems, farmers still have a major impact on crop diversity today. We review several examples of the major impact of man on current diversity. Finally, biodiversity is considered to be an asset for adaptation to current environmental changes. We describe the evolution of pearl millet in West Africa, where average rainfall has decreased over the last forty years. Diversity in cultivated varieties has certainly helped this crop to adapt to climate variation.

A set of 100 chloroplast DNA primer pairs to study population genetics and phylogeny in monocotyledons.

Chloroplast DNA sequences are of great interest for population genetics and phylogenetic studies. However, only a small set of markers are commonly used. Most of them have been designed for amplification in a large range of Angiosperms and are located in the Large Single Copy (LSC). Here we developed a new set of 100 primer pairs optimized for amplification in Monocotyledons. Primer pairs amplify coding (exon) and non-coding regions (intron and intergenic spacer). They span the different chloroplast regions: 72 are located in the LSC, 13 in the Small Single Copy (SSC) and 15 in the Inverted Repeat region (IR). Amplification and sequencing were tested in 13 species of Monocotyledons: Dioscorea abyssinica, D. praehensilis, D. rotundata, D. dumetorum, D. bulbifera, Trichopus sempervirens (Dioscoreaceae), Phoenix canariensis, P. dactylifera, Astrocaryum scopatum, A. murumuru, Ceroxylon echinulatum (Arecaceae), Digitaria excilis and Pennisetum glaucum (Poaceae). The diversity found in Dioscorea, Digitaria and Pennisetum mainly corresponded to Single Nucleotide Polymorphism (SNP) while the diversity found in Arecaceae also comprises Variable Number Tandem Repeat (VNTR). We observed that the most variable loci (rps15-ycf1, rpl32-ccsA, ndhF-rpl32, ndhG-ndhI and ccsA) are located in the SSC. Through the analysis of the genetic structure of a wild-cultivated species complex in Dioscorea, we demonstrated that this new set of primers is of great interest for population genetics and we anticipate that it will also be useful for phylogeny and bar-coding studies.

A weed-crop complex in sorghum: The dynamics of genetic diversity in a traditional farming system.

Despite the major ecological and economic impacts of gene flow between domesticated plants and their wild relatives, many aspects of the process, particularly the relative roles of natural and human selection in facilitating or constraining gene flow, are still poorly understood. We developed a multidisciplinary approach, involving both biologists and social scientists, to investigate the dynamics of genetic diversity of a sorghum weed-crop complex in a village of Duupa farmers in northern Cameroon. Farmers distinguish a gradient from weedy morphotypes (naa baa see, haariya, and genkiya) to domesticated morphotypes; haariya and genkiya have intermediate morphological traits. We investigated the pattern of diversity in this complex using both morphological and genetic data. Our biological results are interpreted in the light of data on farmers' taxonomy and practices such as spatial pattern of planting and plant selection. Both morphological and genetic data are congruent with farmers' taxonomy and confirm the introgressed status of intermediate weedy morphotypes. Farmers actively select against weedy morphotypes, but several practices unconsciously favor gene flow. Furthermore, haariya and genkiya may facilitate introgression between naa baa see and domesticated morphotypes by virtue of their intermediate flowering period and their mode of management by farmers.

Local genetic diversity of sorghum in a village in northern Cameroon: structure and dynamics of landraces.

We present the first study of patterns of genetic diversity of sorghum landraces at the local scale. Understanding landrace diversity aids in deciphering evolutionary forces under domestication, and has applications in the conservation of genetic resources and their use in breeding programs. Duupa farmers in a village in Northern Cameroon distinguished 59 named sorghum taxa, representing 46 landraces. In each field, seeds are sown as a mixture of landraces (mean of 12 landraces per field), giving the potential for extensive gene flow. What level of genetic diversity underlies the great morphological diversity observed among landraces? Given the potential for gene flow, how well defined genetically is each landrace? To answer these questions, we recorded spatial patterns of planting and farmers' perceptions of landraces, and characterized 21 landraces using SSR markers. Analysis using distance and clustering methods grouped the 21 landraces studied into four clusters. These clusters correspond to functionally and ecologically distinct groups of landraces. Within-landrace genetic variation accounted for 30% of total variation. The average F(is) over landraces was 0.68, suggesting high inbreeding within landraces. Differentiation among landraces was substantial and significant (F(st) = 0.36). Historical factors, variation in breeding systems, and farmers' practices all affected patterns of genetic variation. Farmers' practices are key to the maintenance, despite gene flow, of landraces with different combinations of agronomically and ecologically pertinent traits. They must be taken into account in strategies of conservation and use of genetic resources.

Linkage disequilibrium in cultivated grapevine, Vitis vinifera L.

We present here the first study of linkage disequilibrium (LD) in cultivated grapevine, Vitis vinifera L. subsp. vinifera (sativa), an outcrossing highly heterozygous perennial species. Our goal was to characterize the amount and pattern of LD at the scale of a few centiMorgans (cM) between 38 microsatellite loci located on five linkage groups, in order to assess its origin and potential applications. We used a core collection of 141 cultivars representing the diversity of the cultivated compartment. LD was evaluated with both independence tests and multilocus r2, both on raw genotypic and reconstructed haplotypic data. Significant genotypic LD was found only within linkage groups, extending up to 16.8 cM. It appeared not to be influenced by the weak structure of the sample and seemed to be mainly of haplotypic origin. Significant haplotypic LD was found over 30 cM. Both genotypic and haplotypic r2 values declined to around 0.1 within 5-10 cM, suggesting a rather narrow genetic base of the cultivated compartment and limited recombination since domestication events. These first results open up a few application opportunities for association mapping of QTLs and marker assisted selection.