Résumé
Medicago truncatula is a model for investigating legume genetics, including the genetics and evolution of legume–rhizobia symbiosis. We used whole-genome sequence data to identify and characterize sequence polymorphisms and linkage disequilibrium (LD) in a diverse collection of 26 M. truncatula accessions. Our analyses reveal that M. truncatula harbors both higher diversity and less LD than soybean (Glycine max) and exhibits patterns of LD and recombination similar to Arabidopsis thaliana. The population-scaled recombination rate is approximately one-third of the mutation rate, consistent with expectations for a species with a high selfing rate. Linkage disequilibrium, however, is not extensive, and therefore, the low recombination rate is likely not a major constraint to adaptation. Nucleotide diversity in 100-kb windows was negatively correlated with gene density, which is expected if diversity is shaped by selection acting against slightly deleterious mutations. Among putative coding regions, members of four gene families harbor significantly higher diversity than the genome-wide average. Three of these families are involved in resistance against pathogens; one of these families, the nodule-specific, cysteine-rich gene family, is specific to the galegoid legumes and is involved in control of rhizobial differentiation. The more than 3 million SNPs that we detected, approximately one-half of which are present in more than one accession, are a valuable resource for genome-wide association mapping of genes responsible for phenotypic diversity in legumes, especially traits associated with symbiosis and nodulation.
Author SummaryFig. P1.Results of an analysis of nucleotide sequence variability (estimated for 100-kb windows) of chromosome 5 show that (i) nucleotide diversity (θW) of M. truncatula is higher near the centromere (the region that joins the chromosome arms) than the telomere (the tip of the chromosome arm) and (ii) nucleotide diversity is negatively correlated with gene density, which was expected if the removal of variation by selection is an important force shaping patterns of nucleotide diversity.Based on our analyses and the current costs of whole-genome resequencing, a tagged SNP approach for conducting GWAS in M. truncatula does not provide a clear advantage over whole-genome resequencing. In particular, a tagged SNP approach designed to assay all common SNPs detected in our sample would require more than 800,000 tagged SNPs. Moreover, such a strategy would entail substantial bias and impede assaying of low-frequency SNPs. Avoiding these limitations would decrease the probability of identifying potentially misleading synthetic associations (1) and increase the power to correctly identify causal variants and characterize the genetic architecture of complex traits.Linkage disequilibrium (LD) is the nonrandom association between two or more genetic variants that may or may not be in the same chromosomal region. Relative to species that undergo outcrossing, those species that are predominantly self-fertilizing might be expected to have extended LD among polymorphisms (sequence variants) and low rates of effective recombination. Within our sample, however, approximately two-thirds of the SNPs detected were not in complete LD with an adjacent SNP. Moreover, although LD was highly variable (spanning the entire range from absence of to complete LD over distances from 1 to 10 kb), the average LD extended less than 5 kb. At the genome scale, these patterns are very similar to those patterns found in the predominantly self-fertilizing A. thaliana. Interestingly, neither of these species shows patterns consistent with the hypothesis that a self-fertilizing species possesses extensive LD, which would hinder adaptive evolution. However, the patterns in both species support the expectation that the evolutionary transition from outcrossing to self-fertilizing will have a much greater effect on recombination than mutation (5).In multiple species, genomic regions with low levels of recombination harbor less nucleotide variation than those regions with high recombination (4), and we find this pattern in M. truncatula as well (Fig. P1). This phenomenon is consistent with both background selection (i.e., selection against deleterious mutations) and genetic hitchhiking (i.e., linkage to beneficial mutations that are favored by selection) that reduce genetic diversity at linked sites throughout the genome. Two aspects of our data, an excess of rare nonsynonymous (amino acid changing) relative to synonymous (mutations that do not affect the amino acid sequence of a protein) mutations and the absence of a correlation between gene density and rare variants, suggest that the negative correlation between recombination and nucleotide diversity in M. truncatula is primarily because of background selection rather than hitchhiking. Consistent with a limited role for hitchhiking, we found that few regions in the genome exhibited strong signatures of recent selective sweeps.M. truncatula is a predominantly self-fertilizing plant species that serves as a model for investigating the genetics and genome evolution of legumes as well as cooperation between legumes and the bacteria that fix nitrogen when in root nodules and between plants and beneficial fungi. We sequenced an average of 82 million 90-base paired end reads from each of 26 M. truncatula lines. After applying strict quality filters to the sequence information, we identified more than 3 million genome markers known as SNPs, which revealed the nucleotide diversity of this legume to be slightly greater than the diversity of A. thaliana or soybean but lower than the diversity of the highly diverse maize. Moreover, nucleotide diversity was found to be high in classes of genes with well-established roles in defense against pathogens and/or control of differentiation of the symbiotic bacteria involved in nitrogen fixation in the nodules. The high diversity in these gene families may reflect selection for rare genetic variants or reduced selection acting on nonfunctional members of these large gene families.Sequencing the genomes of multiple individuals of a single species, a field known as population genomics, provides the opportunity to extend our understanding of mutation, recombination, and selection in shaping genomic diversity at the species level. Beyond evolutionary processes, the sequencing data from a population sample are important for developing the tools and resources needed for genome-wide association studies (GWAS). The use of GWAS, especially in humans, for identifying genetic variants that influence or have profound effects on complex traits, such as disease susceptibility, height, and yield, remains challenging (1). However, in the case of plant species where data on various traits can be collected in highly replicated experiments with controlled environmental conditions, GWAS seems to be a potentially powerful method for identifying the genes underlying trait variation [e.g., maize (2) and Arabidopsis (3)]. In this study, we used rapid sequencing technology (Illumina next generation DNA sequencing technology) to examine nucleotide diversity in Medicago truncatula, a relative of alfalfa. The patterns of diversity reveal selection acting against most mutations that alter protein sequences, high diversity in genes involved in biotic interactions, and low levels of recombination relative to mutation. Importantly, the sequence information provides a powerful basis for conducting GWAS in M. truncatula.The authors declare no conflict of interest.This article is a PNAS Direct Submission.Data deposition: The sequence reported in this paper has been deposited in the NCBI Sequence Read Archive (accession no. SRP001874).See full research article on page E864 of www.pnas.org.Cite this Author Summary as: PNAS 10.1073/pnas.1104032108.1SP Dickson, K Wang, I Krantz, H Hakonarson, DB Goldstein, Rare variants create synthetic genome-wide associations. PLoS Biol 8, e1000294 (2010).2F Tian, et al., Genome-wide association study of leaf architecture in the maize nested association mapping population. Nat Genet 43, 159–162 (2011).3S Atwell, et al., Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines. Nature 465, 627–631 (2010).4W Stephan, Genetic hitchhiking versus background selection: The controversy and its implications. Philos Trans R Soc Lond B Biol Sci 365, 1245–1253 (2010).5M Nordborg, Linkage disequilibrium, gene trees and selfing: An ancestral recombination graph with partial self-fertilization. Genetics 154, 923–929 (2000).