Cédric Mariac

Diversity of wild and cultivated pearl millet accessions (Pennisetum glaucum [L.] R. Br.) in Niger assessed by microsatellite markers

Genetic diversity of crop species in sub-Sahelian Africa is still poorly documented. Among such crops, pearl millet is one of the most important staple species. In Niger, pearl millet covers more than 65% of the total cultivated area. Analyzing pearl millet genetic diversity, its origin and its dynamics is important for in situ and ex situ germplasm conservation and to increase knowledge useful for breeding programs. We developed new genetic markers and a high-throughput technique for the genetic analysis of pearl millet. Using 25 microsatellite markers, we analyzed genetic diversity in 46 wild and 421 cultivated accessions of pearl millet in Niger. We showed a significantly lower number of alleles and lower gene diversity in cultivated pearl millet accessions than in wild accessions. This result contrasts with a previous study using iso-enzyme markers showing similar genetic diversity between cultivated and wild pearl millet populations. We found a strong differentiation between the cultivated and wild groups in Niger. Analyses of introgressions between cultivated and wild accessions showed modest but statistically supported evidence of introgressions. Wild accessions in the central region of Niger showed introgressions of cultivated alleles. Accessions of cultivated pearl millet showed introgressions of wild alleles in the western, central, and eastern parts of Niger.

Association Studies Identify Natural Variation at PHYC Linked to Flowering Time and Morphological Variation in Pearl Millet

The identification of genes selected during and after plant domestication is an important research topic to enhance knowledge on adaptative evolution. Adaptation to different climates was a key factor in the spread of domesticated crops. We conducted a study to identify genes responsible for these adaptations in pearl millet and developed an association framework to identify genetic variations associated with the phenotype in this species. A set of 90 inbred lines genotyped using microsatellite loci and AFLP markers was used. The population structure was assessed using two different Bayesian approaches that allow inbreeding or not. Association studies were performed using a linear mixed model considering both the population structure and familial relationships between inbred lines. We assessed the ability of the method to limit the number of false positive associations on the basis of the two different Bayesian methods, the number of populations considered and different morphological traits while also assessing the power of the methodology to detect given additive effects. Finally, we applied this methodology to a set of eight pearl millet genes homologous to cereal flowering pathway genes. We found significant associations between several polymorphisms of the pearl millet PHYC gene and flowering time, spike length, and stem diameter in the inbred line panel. To validate this association, we performed a second association analysis in a different set of pearl millet individuals from Niger. We confirmed a significant association between genetic variation in this gene and these characters.

Genetic Nature of Yams (Dioscorea sp.) Domesticated by Farmers in Benin (West Africa)

Abstract‘Domestication’ is a traditional farmers’ practice reported for yams (Dioscorea sp.) in Benin (West Africa). It involves introducing ‘spontaneous’ (naturally occurring) yams, supposedly wild (D. abyssinica and D. praehensilis), in varieties of the D. cayenensis–D. rotundata cultivated species complex. In this study, we established the genetic nature of ‘predomesticated’ yam plants using the amplified fragment length polymorphism (AFLP) technique. A total of 213 accessions, consisting of 32 predomesticated yams, 70 D. cayenensis–D. rotundata, 86 D. abyssinica and 25 D. praehensilis yams were analysed. Using 91 AFLP markers, three groups of accessions were distinguished, broadly corresponding to the above botanical species. Of the 32 predomesticated accessions, 16% were clustered with D. praehensilis, 37% with D. abyssinica and the remaining 47% with D. cayenensis–D. rotundata. These results demonstrated the use of wild plants by farmers in their domestication process, and suggested that plants derived from intervarietal and interspecific hybridisation may also be subject to this process. This study has shown that through domestication farmers influence and increase the genetic diversity in yam by using sexual reproduction of wild and possibly cultivated yams.

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.

Selection for Earlier Flowering Crop Associated with Climatic Variations in the Sahel

Climate changes will have an impact on food production and will require costly adaptive responses. Adapting to a changing environment will be particularly challenging in sub-Saharan Africa where climate change is expected to have a major impact. However, one important phenomenon that is often overlooked and is poorly documented is the ability of agro-systems to rapidly adapt to environmental variations. Such an adaptation could proceed by the adoption of new varieties or by the adaptation of varieties to a changing environment. In this study, we analyzed these two processes in one of the driest agro-ecosystems in Africa, the Sahel. We performed a detailed study in Niger where pearl millet is the main crop and covers 65% of the cultivated area. To assess how the agro-system is responding to recent recurrent drought, we analyzed samples of pearl millet landraces collected in the same villages in 1976 and 2003 throughout the entire cultivated area of Niger. We studied phenological and morphological differences in the 1976 and 2003 collections by comparing them over three cropping seasons in a common garden experiment. We found no major changes in the main cultivated varieties or in their genetic diversity. However, we observed a significant shift in adaptive traits. Compared to the 1976 samples, samples collected in 2003 displayed a shorter lifecycle, and a reduction in plant and spike size. We also found that an early flowering allele at the PHYC locus increased in frequency between 1976 and 2003. The increase exceeded the effect of drift and sampling, suggesting a direct effect of selection for earliness on this gene. We conclude that recurrent drought can lead to selection for earlier flowering in a major Sahelian crop. Surprisingly, these results suggest that diffusion of crop varieties is not the main driver of short term adaptation to climatic variation.

Genetic basis of pearl millet adaptation along an environmental gradient investigated by a combination of genome scan and association mapping

Identifying the molecular bases of adaptation is a key issue in evolutionary biology. Genome scan is an efficient approach for identifying important molecular variation involved in adaptation. Association mapping also offers an opportunity to gain insight into genotype–phenotype relationships. Using these two approaches coupled with environmental data should help to come up with a refined picture of the evolutionary process underlying adaptation. In this study, we first conducted a selection scan analysis on a transcription factor gene family. We focused on the MADS‐box gene family, a gene family which plays a crucial role in vegetative and flower development. Twenty‐one pearl millet populations were sampled along an environmental gradient in West Africa. We identified one gene, i.e. PgMADS11, using Bayesian analysis to detect selection signatures. Polymorphism at this gene was also associated with flowering time variation in an association mapping framework. Finally, we found that PgMADS11 allele frequencies were closely associated with annual rainfall. Overall, we determined an efficient way to detect functional polymorphisms associated with climate variation in non‐model plants by combining genome scan and association mapping. These results should help monitor the impact of recent climatic changes on plant adaptation.

Evolutionary History of Pearl Millet (Pennisetum glaucum [L.] R. Br.) and Selection on Flowering Genes since Its Domestication

The plant domestication process is associated with considerable modifications of plant phenotype. The identification of the genetic basis of this adaptation is of great interest for evolutionary biology. One of the methods used to identify such genes is the detection of signatures of selection. However, domestication is generally associated with major demographic effects. It is therefore crucial to disentangle the effects of demography and selection on diversity. In this study, we investigated selection in a flowering time pathway during domestication of pearl millet. We first used a random set of 20 genes to model pearl millet domestication using approximate Bayesian computation. This analysis showed that a model with exponential growth and wild-cultivated gene flow was well supported by our data set. Under this model, the domestication date of pearl millet is estimated at around 4,800 years ago. We assessed selection in 15 pearl millet DNA sequences homologous to flowering time genes and showed that these genes underwent selection more frequently than expected. We highlighted significant signatures of selection in six pearl millet flowering time genes associated with domestication or improvement of pearl millet. Moreover, higher deviations from neutrality were found for circadian clock-associated genes. Our study provides new insights into the domestication process of pearl millet and shows that a category of genes of the flowering pathway were preferentially selected during pearl millet domestication.

Role of seed flow on the pattern and dynamics of pearl millet (Pennisetum glaucum [L.] R. Br.) genetic diversity assessed by AFLP markers: a study in south-western Niger

We studied the regional genetic diversity and seed exchange dynamics of pearl millet landraces in south-western Niger. The genetic study was based on AFLP markers. We found significant genetic differentiation between landraces in different geographical areas of south-western Niger. However, the degree of differentiation was low insofar as only 1.9% of the total molecular diversity was due to regional differentiation, suggesting a relatively high gene flow. Anthropologic studies on farming practices have suggested that seed exchanges between farmers on a large geographical scale probably make a considerable contribution to this result. In order to test this hypothesis, the effects of seed exchange on the genetic diversity of landraces was analyzed on seed samples from two distant villages in contrasting areas of south-western Niger. Seeds imported by farmers into the southern village of Sina Koara did not differ significantly from locally grown landraces. By contrast, in the northern village of Alzou, several samples were genetically different from locally grown landraces and closer to southern accessions. These data suggest that the seed flow is preferentially from south to north, i.e. from an area with more favorable rainfall conditions. The potential consequences for the genetic diversity and adaptation of northern pearl millet landraces are discussed.

Genetic diversity analysis of yam cultivars (Dioscorea rotundata Poir.) in Benin using simple sequence repeat (SSR) markers

Abstract Guinea yam (Dioscorea rotundata Poir.) is a dioecious vegetatively propagated tuber crop. It iswidely cultivated by traditional techniques in West Africa, its area of origin. The genetic diver-sity of 146 accessions from Benin was analysed using 10 polymorphic simple sequence repeat(SSR) nuclear markers and agromorphological traits. An average of 8.4 alleles per locus wasdetected. The mean heterozygosity was 0.57 and the mean polymorphism information content(PIC) for polymorphic markers was 0.51. Some cultivars (23%) were found to have an identicalgenotype for the 10 markers. The structure of the genetic diversity observed in Benin is theresult of farmers’ crop management practices and their know-how. The cultivar diversityhad a geographical component. We also noted major differentiation between early and latecultivars, with higher diversity in the early ones. Cultivars from northern Benin and early cul-tivars had the greatest allelic richness. SSR markers proved to be powerful tools for fingerprint-ing each cultivar and analysing their genetic relationships. The results of this study could beuseful for defining a strategy for the conservation of genetic diversity in yams.

Intra-individual polymorphism in chloroplasts from NGS data: where does it come from and how to handle it?

Next‐generation sequencing allows access to a large quantity of genomic data. In plants, several studies used whole chloroplast genome sequences for inferring phylogeography or phylogeny. Even though the chloroplast is a haploid organelle, NGS plastome data identified a nonnegligible number of intra‐individual polymorphic SNPs. Such observations could have several causes such as sequencing errors, the presence of heteroplasmy or transfer of chloroplast sequences in the nuclear and mitochondrial genomes. The occurrence of allelic diversity has practical important impacts on the identification of diversity, the analysis of the chloroplast data and beyond that, significant evolutionary questions. In this study, we show that the observed intra‐individual polymorphism of chloroplast sequence data is probably the result of plastid DNA transferred into the mitochondrial and/or the nuclear genomes. We further assess nine different bioinformatics pipelines’ error rates for SNP and genotypes calling using SNPs identified in Sanger sequencing. Specific pipelines are adequate to deal with this issue, optimizing both specificity and sensitivity. Our results will allow a proper use of whole chloroplast NGS sequence and will allow a better handling of NGS chloroplast sequence diversity.