Nora Scarcelli

A multiparent advanced generation inter-cross to fine-map quantitative traits in Arabidopsis thaliana

Identifying natural allelic variation that underlies quantitative trait variation remains a fundamental problem in genetics. Most studies have employed either simple synthetic populations with restricted allelic variation or performed association mapping on a sample of naturally occurring haplotypes. Both of these approaches have some limitations, therefore alternative resources for the genetic dissection of complex traits continue to be sought. Here we describe one such alternative, the Multiparent Advanced Generation Inter-Cross (MAGIC). This approach is expected to improve the precision with which QTL can be mapped, improving the outlook for QTL cloning. Here, we present the first panel of MAGIC lines developed: a set of 527 recombinant inbred lines (RILs) descended from a heterogeneous stock of 19 intermated accessions of the plant Arabidopsis thaliana. These lines and the 19 founders were genotyped with 1,260 single nucleotide polymorphisms and phenotyped for development-related traits. Analytical methods were developed to fine-map quantitative trait loci (QTL) in the MAGIC lines by reconstructing the genome of each line as a mosaic of the founders. We show by simulation that QTL explaining 10% of the phenotypic variance will be detected in most situations with an average mapping error of about 300 kb, and that if the number of lines were doubled the mapping error would be under 200 kb. We also show how the power to detect a QTL and the mapping accuracy vary, depending on QTL location. We demonstrate the utility of this new mapping population by mapping several known QTL with high precision and by finding novel QTL for germination data and bolting time. Our results provide strong support for similar ongoing efforts to produce MAGIC lines in other organisms.

Antagonistic pleiotropic effects reduce the potential adaptive value of the FRIGIDA locus

Although the occurrence of epistasis and pleiotropy is widely accepted at the molecular level, its effect on the adaptive value of fitness-related genes is rarely investigated in plants. Knowledge of these features of a gene is critical to understand the molecular basis of adaptive evolution. Here we investigate the importance of pleiotropy and epistasis in determining the adaptive value of a candidate gene using the gene FRI (FRIGIDA), which is thought to be the major gene controlling flowering time variation in Arabidopsis thaliana. The effect of FRI on flowering time was analyzed in an outbred population created by randomly mating 19 natural accessions of A. thaliana. This unique population allows the estimation of FRI effects independent of any linkage association with other loci due to demographic processes or to coadapted genes. It also allows for the estimation of pleiotropic effects of FRI on fitness and inflorescence architecture. We found that FRI explains less variation in flowering time than previously observed among natural accessions, and interacts epistatically with the FLC locus. Although early flowering plants produce more fruits under spring conditions, and nonfunctional alleles of FRI were associated with early flowering, variation at FRI was not associated with fitness. We show that nonfunctional FRI alleles have negative pleiotropic effects on fitness by reducing the numbers of nodes and branches on the inflorescence. We propose that these antagonistic pleiotropic effects reduce the adaptive value of FRI, and helps explain the maintenance of alternative life history strategies across natural populations of A. thaliana.

Farmers’ use of wild relative and sexual reproduction in a vegetatively propagated crop. The case of yam in Benin

The impact of traditional farmers’ management on genetic diversity of vegetatively propagated crops is poorly documented. In this study, we analysed the impact of ennoblement of spontaneous yams, an original traditional farmers’ practice, on the genetic diversity of yam (Dioscorea sp.) in Benin. We used 11 microsatellite markers on yam tubers from a small village in northern Benin and demonstrated that wild × cultivated hybrids are spontaneously formed. Many of the spontaneous yams collected by farmers from surrounding savannah areas for ennoblement were shown to be of wild and hybrid genotypes. Moreover, we demonstrated that some yam varieties have a wild or hybrid signature. Lastly, we performed a broader ranging genetic analysis on yam material from throughout Benin and showed that this practice is used in different ecological and ethno‐linguistic regions. Through this practice, farmers create new varieties with new genetic combinations via sexual reproduction of wild and cultivated yams. This system, whereby a sexual cycle and asexual propagation are mixed, ensures potential large‐scale cultivation of the best genotypes while preserving the potential for future adaptation.

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.

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.

Segregation patterns of isozyme loci and microsatellite markers show the diploidy of African yam Dioscorea rotundata (2n=40)

The cultivated yam species Dioscorea rotundata (2n=40) has been considered by most authors as a tetraploid species with a basic chromosome number of ten. In this paper, we analysed the segregation of two isozyme loci and six microsatellite markers in the progeny of a self-fertilised monoecious plant. For the eight markers, segregation patterns could be explained by only two genetic models: diploidy or tetraploidy with two null alleles. Given the nature of studied markers, the most parsimonious hypothesis was that the parental plant was diploid. These results, data from a diversity survey and results of other authors led to the conclusion that D. rotundata is a diploid species.

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.

Standing genetic variation in FRIGIDA mediates experimental evolution of flowering time in Arabidopsis

The role of standing genetic variation in adaptive evolution remains unclear. Although there has been much progress in identifying candidate genes that underlie adaptive traits, we still lack direct evidence that natural allelic variation in these genes can actually mediate adaptive evolution. In this study, we investigate the role of natural allelic variation in two candidate flowering time genes, in response to selection for early flowering in Arabidopsis thaliana: FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). We performed artificial selection for early flowering under ‘spring‐’ and ‘winter‐annual’ growth conditions using an outbred population of A. thaliana produced by intermating 19 natural accessions. FRI and FLC are involved in A. thalianas response to winter conditions, and nonfunctional and weak alleles at these loci are know to reduce flowering time, particularly under spring‐annual conditions. Our results provide direct evidence that natural allelic variation in FRI can provide rapid and predictable adaptive evolution in flowering time under spring‐annual conditions. We observed a strong response to selection, in terms of reducing flowering time, in both growth conditions (~2 standard deviation reduction). Concomitantly, the frequency of functional FRI alleles under spring‐annual conditions was reduced by 68%, in agreement with predicted changes. No significant changes in allele frequencies were observed in FRI in the winter‐annual growth condition or in FLC for either growth conditions. These results indicate that changes in flowering time are mediated by different genetic factors under spring‐ and winter‐annual growth conditions, and that other loci must also be contributing to the response to selection.

Pleiotropic effects of environment‐specific adaptation in Arabidopsis thaliana

Local adaptation may be important for the preservation of genetic diversity and the promotion of speciation. However, local adaptation may also constrain establishment in different environments. The consequences of local adaptation depend strongly on the pleiotropic effects of the genes involved in adaptation. Here, we investigated the pleiotropic effects of the genetic response to selection in outbred lines of Arabidopsis artificially selected to flower earlier under both winter- and spring-annual simulated conditions. The consequences of adaptation were evaluated by reciprocally transplanting selected and control lines between the two conditions. Selected lines always flower earlier than their controls, independent of growing conditions. However, selected lines, growing in the same condition in which they were selected, flower earlier than plants selected in the alternative environment. Plants selected to flower earlier in spring produce more fruits than controls when growing in the spring, and less fruits when growing in the winter; indicating that local adaptation has negative pleiotropic effects in another environment. Our results indicate that local adaptation can arise even when selection targets the same trait in the same direction. Furthermore, it suggests that adaptation under the two different environments can generate fitness trade-offs that can maintain genetic variation for flowering time.