Christopher Sauvage

Genome-Wide Association in Tomato Reveals 44 Candidate Loci for Fruit Metabolic Traits

Genome-wide association sheds light on the genetic architecture of metabolic traits underlying fruit quality in tomato. Genome-wide association studies have been successful in identifying genes involved in polygenic traits and are valuable for crop improvement. Tomato (Solanum lycopersicum) is a major crop and is highly appreciated worldwide for its health value. We used a core collection of 163 tomato accessions composed of S. lycopersicum, S. lycopersicum var cerasiforme, and Solanum pimpinellifolium to map loci controlling variation in fruit metabolites. Fruits were phenotyped for a broad range of metabolites, including amino acids, sugars, and ascorbate. In parallel, the accessions were genotyped with 5,995 single-nucleotide polymorphism markers spread over the whole genome. Genome-wide association analysis was conducted on a large set of metabolic traits that were stable over 2 years using a multilocus mixed model as a general method for mapping complex traits in structured populations and applied to tomato. We detected a total of 44 loci that were significantly associated with a total of 19 traits, including sucrose, ascorbate, malate, and citrate levels. These results not only provide a list of candidate loci to be functionally validated but also a powerful analytical approach for finding genetic variants that can be directly used for crop improvement and deciphering the genetic architecture of complex traits.

A cytochrome P450 regulates a domestication trait in cultivated tomato.

Significance This study reports the cloning of a tomato gene, SlKLUH, that controls fruit mass by increased cell layers and delayed fruit ripening. In addition, we identified a potential regulatory SNP in the promoter of SlKLUH that is significantly associated with the fruit mass. Altogether, our study encompasses several genetic analyses, as well as association mapping, plant transformation experiments, and phenotypic evaluations to offer insights into the molecular basis of the regulation of tomato fruit mass, a critical trait in the domestication of fruit and vegetable crops. Domestication of crop plants had effects on human lifestyle and agriculture. However, little is known about the underlying molecular mechanisms accompanying the changes in fruit appearance as a consequence of selection by early farmers. We report the fine mapping and cloning of a tomato (Solanum lycopersicum) fruit mass gene encoding the ortholog of KLUH, SlKLUH, a P450 enzyme of the CYP78A subfamily. The increase in fruit mass is predominantly the result of enlarged pericarp and septum tissues caused by increased cell number in the large fruited lines. SlKLUH also modulates plant architecture by regulating number and length of the side shoots, and ripening time, and these effects are particularly strong in plants that transgenically down-regulate SlKLUH expression carrying fruits of a dramatically reduced mass. Association mapping followed by segregation analyses revealed that a single nucleotide polymorphism in the promoter of the gene is highly associated with fruit mass. This single polymorphism may potentially underlie a regulatory mutation resulting in increased SlKLUH expression concomitant with increased fruit mass. Our findings suggest that the allele giving rise to large fruit arose in the early domesticates of tomato and becoming progressively more abundant upon further selections. We also detected association of fruit weight with CaKLUH in chile pepper (Capsicum annuum) suggesting that selection of the orthologous gene may have occurred independently in a separate domestication event. Altogether, our findings shed light on the molecular basis of fruit mass, a key domestication trait in tomato and other fruit and vegetable crops.

Genomic variation in tomato, from wild ancestors to contemporary breeding accessions

BackgroundDomestication modifies the genomic variation of species. Quantifying this variation provides insights into the domestication process, facilitates the management of resources used by breeders and germplasm centers, and enables the design of experiments to associate traits with genes. We described and analyzed the genetic diversity of 1,008 tomato accessions including Solanum lycopersicum var. lycopersicum (SLL), S. lycopersicum var. cerasiforme (SLC), and S. pimpinellifolium (SP) that were genotyped using 7,720 SNPs. Additionally, we explored the allelic frequency of six loci affecting fruit weight and shape to infer patterns of selection.ResultsOur results revealed a pattern of variation that strongly supported a two-step domestication process, occasional hybridization in the wild, and differentiation through human selection. These interpretations were consistent with the observed allele frequencies for the six loci affecting fruit weight and shape. Fruit weight was strongly selected in SLC in the Andean region of Ecuador and Northern Peru prior to the domestication of tomato in Mesoamerica. Alleles affecting fruit shape were differentially selected among SLL genetic subgroups. Our results also clarified the biological status of SLC. True SLC was phylogenetically positioned between SP and SLL and its fruit morphology was diverse. SLC and “cherry tomato” are not synonymous terms. The morphologically-based term “cherry tomato” included some SLC, contemporary varieties, as well as many admixtures between SP and SLL. Contemporary SLL showed a moderate increase in nucleotide diversity, when compared with vintage groups.ConclusionsThis study presents a broad and detailed representation of the genomic variation in tomato. Tomato domestication seems to have followed a two step-process; a first domestication in South America and a second step in Mesoamerica. The distribution of fruit weight and shape alleles supports that domestication of SLC occurred in the Andean region. Our results also clarify the biological status of SLC as true phylogenetic group within tomato. We detect Ecuadorian and Peruvian accessions that may represent a pool of unexplored variation that could be of interest for crop improvement.

Dissecting quantitative trait variation in the resequencing era: complementarity of bi-parental, multi-parental and association panels.

Quantitative trait loci (QTL) have been identified using traditional linkage mapping and positional cloning identified several QTLs. However linkage mapping is limited to the analysis of traits differing between two lines and the impact of the genetic background on QTL effect has been underlined. Genome-wide association studies (GWAs) were proposed to circumvent these limitations. In tomato, we have shown that GWAs is possible, using the admixed nature of cherry tomato genomes that reduces the impact of population structure. Nevertheless, GWAs success might be limited due to the low decay of linkage disequilibrium, which varies along the genome in this species. Multi-parent advanced generation intercross (MAGIC) populations offer an alternative to traditional linkage and GWAs by increasing the precision of QTL mapping. We have developed a MAGIC population by crossing eight tomato lines whose genomes were resequenced. We showed the potential of the MAGIC population when coupled with whole genome sequencing to detect candidate single nucleotide polymorphisms (SNPs) underlying the QTLs. QTLs for fruit quality traits were mapped and related to the variations detected at the genome sequence and expression levels. The advantages and limitations of the three types of population, in the context of the available genome sequence and resequencing facilities, are discussed.

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/.

Genes involved in floral meristem in tomato exhibit drastically reduced genetic diversity and signature of selection

BackgroundDomestication and selection of crops have notably reshaped fruit morphology. With its large phenotypic diversity, tomato (Solanum lycopersicum) illustrates this evolutive trend. Genes involved in flower meristem development are known to regulate also fruit morphology. To decipher the genetic variation underlying tomato fruit morphology, we assessed the nucleotide diversity and selection footprints of candidate genes involved in flower and fruit development and performed genome-wide association studies.ResultsThirty candidate genes were selected according to their similarity with genes involved in meristem development or their known causal function in Arabidopsis thaliana. In tomato, these genes and flanking regions were sequenced in a core collection of 96 accessions (including cultivated, cherry-type and wild relative accessions) maximizing the molecular diversity, using the Roche 454 technology. A total amount of 17 Mb was sequenced allowing the discovery of 6,106 single nucleotide polymorphisms (SNPs). The annotation of the 30 gene regions identified 231 exons carrying 517 SNPs. Subsequently, the nucleotide diversity (π) and the neutral evolution of each region were compared against genome-wide values within the collection, using a SNP array carrying 7,667 SNPs mainly distributed in coding sequences.About half of the genes revealed footprints of selection and polymorphisms putatively involved in fruit size variation by showing negative Tajimas D and nucleotide diversity reduction in cultivated tomato compared to its wild relative. Among the candidates, FW2.2 and BAM1 sequences revealed selection footprints within their promoter regions suggesting their potential involvement in their regulation. Two associations co-localized with previously identified loci: LC (locule number) and Ovate (fruit shape).ConclusionCompared to whole genome genotypic data, a drastic reduction of nucleotide diversity was shown for several candidate genes. Strong selection patterns were identified in 15 candidates highlighting the critical role of meristem maintenance genes as well as the impact of domestication on candidates. The study highlighted a set of polymorphisms putatively important in the evolution of these genes.

Evolutionary forces affecting synonymous variations in plant genomes

Base composition is highly variable among and within plant genomes, especially at third codon positions, ranging from GC-poor and homogeneous species to GC-rich and highly heterogeneous ones (particularly Monocots). Consequently, synonymous codon usage is biased in most species, even when base composition is relatively homogeneous. The causes of these variations are still under debate, with three main forces being possibly involved: mutational bias, selection and GC-biased gene conversion (gBGC). So far, both selection and gBGC have been detected in some species but how their relative strength varies among and within species remains unclear. Population genetics approaches allow to jointly estimating the intensity of selection, gBGC and mutational bias. We extended a recently developed method and applied it to a large population genomic dataset based on transcriptome sequencing of 11 angiosperm species spread across the phylogeny. We found that at synonymous positions, base composition is far from mutation-drift equilibrium in most genomes and that gBGC is a widespread and stronger process than selection. gBGC could strongly contribute to base composition variation among plant species, implying that it should be taken into account in plant genome analyses, especially for GC-rich ones.

Eggplant Resistance to the Ralstonia solanacearum Species Complex Involves Both Broad-Spectrum and Strain-Specific Quantitative Trait Loci

Bacterial wilt (BW) is a major disease of solanaceous crops caused by the Ralstonia solanacearum species complex (RSSC). Strains are grouped into five phylotypes (I, IIA, IIB, III, and IV). Varietal resistance is the most sustainable strategy for managing BW. Nevertheless, breeding to improve cultivar resistance has been limited by the pathogen’s extensive genetic diversity. Identifying the genetic bases of specific and non-specific resistance is a prerequisite to breed improvement. A major gene (ERs1) was previously mapped in eggplant (Solanum melongena L.) using an intraspecific population of recombinant inbred lines derived from the cross of susceptible MM738 (S) × resistant AG91-25 (R). ERs1 was originally found to control three strains from phylotype I, while being totally ineffective against a virulent strain from the same phylotype. We tested this population against four additional RSSC strains, representing phylotypes I, IIA, IIB, and III in order to clarify the action spectrum of ERs1. We recorded wilting symptoms and bacterial stem colonization under controlled artificial inoculation. We constructed a high-density genetic map of the population using single nucleotide polymorphisms (SNPs) developed from genotyping-by-sequencing and added 168 molecular markers [amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), and sequence-related amplified polymorphisms (SRAPs)] developed previously. The new linkage map based on a total of 1,035 markers was anchored on eggplant, tomato, and potato genomes. Quantitative trait locus (QTL) mapping for resistance against a total of eight RSSC strains resulted in the detection of one major phylotype-specific QTL and two broad-spectrum QTLs. The major QTL, which specifically controls three phylotype I strains, was located at the bottom of chromosome 9 and corresponded to the previously identified major gene ERs1. Five candidate R-genes were underlying this QTL, with different alleles between the parents. The two other QTLs detected on chromosomes 2 and 5 were found to be associated with partial resistance to strains of phylotypes I, IIA, III and strains of phylotypes IIA and III, respectively. Markers closely linked to these three QTLs will be crucial for breeding eggplant with broad-spectrum resistance to BW. Furthermore, our study provides an important contribution to the molecular characterization of ERs1, which was initially considered to be a major resistance gene.

A new eIF4E1 allele characterized by RNAseq data mining is associated with resistance to potato virus Y in tomato albeit with a low durability.

Allele mining on susceptibility factors offers opportunities to find new sources of resistance among crop wild relatives for breeding purposes. As a proof of concept, we used available RNAseq data to investigate polymorphisms among the four tomato genes encoding translation initiation factors [eIF4E1 and eIF4E2, eIFiso4E and the related gene new cap-binding protein(nCBP)] to look for new potential resistance alleles to potyviruses. By analysing polymorphism among RNAseq data obtained for 20 tomato accessions, 10 belonging to the cultivated type Solanum lycopersicum and 10 belonging to the closest related wild species Solanum pimpinellifolium, we isolated one new eIF4E1 allele, in the S. pimpinellifolium LA0411 accession, which encodes a potential new resistance allele, mainly due to a polymorphism associated with an amino acid change within eIF4E1 region II. We confirmed that this new allele, pot12, is indeed associated with resistance to potato virus Y, although with a restricted resistance spectrum and a very low durability potential. This suggests that mutations occurring in eIF4E region II only may not be sufficient to provide efficient and durable resistance in plants. However, our study emphasizes the opportunity brought by RNAseq data to mine for new resistance alleles. Moreover, this approach could be extended to seek for putative new resistance alleles by screening for variant forms of susceptibility genes encoding plant host proteins known to interact with viral proteins.

Domestication rewired gene expression and nucleotide diversity patterns in tomato

Plant domestication has led to considerable phenotypic modifications from wild species to modern varieties. However, although changes in key traits have been well documented, less is known about the underlying molecular mechanisms, such as the reduction of molecular diversity or global gene co-expression patterns. In this study, we used a combination of gene expression and population genetics in wild and crop tomato to decipher the footprints of domestication. We found a set of 1729 differentially expressed genes (DEG) between the two genetic groups, belonging to 17 clusters of co-expressed DEG, suggesting that domestication affected not only individual genes but also regulatory networks. Five co-expression clusters were enriched in functional terms involving carbohydrate metabolism or epigenetic regulation of gene expression. We detected differences in nucleotide diversity between the crop and wild groups specific to DEG. Our study provides an extensive profiling of the rewiring of gene co-expression induced by the domestication syndrome in one of the main crop species.