Correlative single-cell hard X-ray computed tomography and X-ray fluorescence imaging.

X-ray computed tomography (XCT) and X-ray fluorescence (XRF) imaging are two non-invasive imaging techniques to study cellular structures and chemical element distributions, respectively. However, correlative X-ray computed tomography and fluorescence imaging for the same cell have yet to be routinely realized due to challenges in sample preparation and X-ray radiation damage. Here we report an integrated experimental and computational workflow for achieving correlative multi-modality X-ray imaging of a single cell. The method consists of the preparation of radiation-resistant single-cell samples using live-cell imaging-assisted chemical fixation and freeze-drying procedures, targeting and labeling cells for correlative XCT and XRF measurement, and computational reconstruction of the correlative and multi-modality images. With XCT, cellular structures including the overall structure and intracellular organelles are visualized, while XRF imaging reveals the distribution of multiple chemical elements within the same cell. Our correlative method demonstrates the feasibility and broad applicability of using X-rays to understand cellular structures and the roles of chemical elements and related proteins in signaling and other biological processes.

Flavonoid Accumulation Varies in Medicago truncatula in Response to Mercury Stress.

Mercury (Hg) contamination is increasing worldwide in both wild ecosystems and agricultural soils due to natural processes, but mostly to anthropic activities. The molecular mechanisms involved in Hg toxicity and tolerance in plants have been extensively studied; however, the role of flavonoids in response to Hg stress remains to be investigated. We conducted a metabolomic study to analyze the changes induced at the secondary metabolite level in three Hg-tolerant and one Hg-sensitive Medicago truncatula cultivars. A total of 46 flavonoid compounds, classified into five different flavonoid families: anthocyanidins, flavones, isoflavones, pterocarpan flavonoids, and flavanones, along with their respective glycoconjugate derivatives, were identified in leaf and root tissues. The synthesis of free isoflavones, followed by monoglycosylation and further malonylation was shown to be characteristic of root samples, whereas higher glycosylation, followed by further acylation with coumaric and ferulic acid was characteristic of leaf tissues. While minor changes were observed in leaves, significant quantitative changes could be observed in roots upon Hg treatment. Some flavonoids were strongly upregulated in roots, including malonylglucosides of biochanin A, formononetin and medicarpin, and aglycones biochanin, daidzein, and irisolidone. Hg tolerance appeared to be mainly associated to the accumulation of formononetin MalGlc, tricin GlcAGlcA, and afrormosin Glc II in leaves, whereas aglycone accumulation was associated with tolerance to Hg stress in roots. The results evidence the alteration of the flavonoid metabolic profile and their glycosylation processes in response to Hg stress. However, notable differences existed between varieties, both in the basal metabolic profile and in the response to treatment with Hg. Overall, we observed an increase in flavonoid production in response to Hg stress, and Hg tolerance appeared to be associated to a characteristic glycosylation pattern in roots, associated with the accumulation of aglycones and monoglycosylated flavonoids. The findings are discussed in the context of the flavonoid biosynthetic pathway to provide a better understanding of the role of these secondary metabolites in the response and tolerance to Hg stress in M. truncatula.

The genome of Shorea leprosula (Dipterocarpaceae) highlights the ecological relevance of drought in aseasonal tropical rainforests.

Hyperdiverse tropical rainforests, such as the aseasonal forests in Southeast Asia, are supported by high annual rainfall. Its canopy is dominated by the species-rich tree family of Dipterocarpaceae (Asian dipterocarps), which has both ecological (e.g., supports flora and fauna) and economical (e.g., timber production) importance. Recent ecological studies suggested that rare irregular drought events may be an environmental stress and signal for the tropical trees. We assembled the genome of a widespread but near threatened dipterocarp, Shorea leprosula, and analyzed the transcriptome sequences of ten dipterocarp species representing seven genera. Comparative genomic and molecular dating analyses suggested a whole-genome duplication close to the Cretaceous-Paleogene extinction event followed by the diversification of major dipterocarp lineages (i.e. Dipterocarpoideae). Interestingly, the retained duplicated genes were enriched for genes upregulated by no-irrigation treatment. These findings provide molecular support for the relevance of drought for tropical trees despite the lack of an annual dry season.

Multiple Wheat Genomes Reveal Novel Gli-2 Sublocus Location and Variation of Celiac Disease Epitopes in Duplicated α-Gliadin Genes.

The seed protein α-gliadin is a major component of wheat flour and causes gluten-related diseases. However, due to the complexity of this multigene family with a genome structure composed of dozens of copies derived from tandem and genome duplications, little was known about the variation between accessions, and thus little effort has been made to explicitly target α-gliadin for bread wheat breeding. Here, we analyzed genomic variation in α-gliadins across 11 recently published chromosome-scale assemblies of hexaploid wheat, with validation using long-read data. We unexpectedly found that the Gli-B2 locus is not a single contiguous locus but is composed of two subloci, suggesting the possibility of recombination between the two during breeding. We confirmed that the number of immunogenic epitopes among 11 accessions varied. The D subgenome of a European spelt line also contained epitopes, in agreement with its hybridization history. Evolutionary analysis identified amino acid sites under diversifying selection, suggesting their functional importance. The analysis opens the way for improved grain quality and safety through wheat breeding.

Genome-Wide Association Study Reveals Complex Genetic Architecture of Cadmium and Mercury Accumulation and Tolerance Traits in Medicago truncatula.

Heavy metals are an increasing problem due to contamination from human sources that and can enter the food chain by being taken up by plants. Understanding the genetic basis of accumulation and tolerance in plants is important for reducing the uptake of toxic metals in crops and crop relatives, as well as for removing heavy metals from soils by means of phytoremediation. Following exposure of Medicago truncatula seedlings to cadmium (Cd) and mercury (Hg), we conducted a genome-wide association study using relative root growth (RRG) and leaf accumulation measurements. Cd and Hg accumulation and RRG had heritability ranging 0.44 - 0.72 indicating high genetic diversity for these traits. The Cd and Hg trait associations were broadly distributed throughout the genome, indicated the traits are polygenic and involve several quantitative loci. For all traits, candidate genes included several membrane associated ATP-binding cassette transporters, P-type ATPase transporters, oxidative stress response genes, and stress related UDP-glycosyltransferases. The P-type ATPase transporters and ATP-binding cassette protein-families have roles in vacuole transport of heavy metals, and our findings support their wide use in physiological plant responses to heavy metals and abiotic stresses. We also found associations between Cd RRG with the genes CAX3 and PDR3, two linked adjacent genes, and leaf accumulation of Hg associated with the genes NRAMP6 and CAX9. When plant genotypes with the most extreme phenotypes were compared, we found significant divergence in genomic regions using population genomics methods that contained metal transport and stress response gene ontologies. Several of these genomic regions show high linkage disequilibrium (LD) among candidate genes suggesting they have evolved together. Minor allele frequency (MAF) and effect size of the most significant SNPs was negatively correlated with large effect alleles being most rare. This is consistent with purifying selection against alleles that increase toxicity and abiotic stress. Conversely, the alleles with large affect that had higher frequencies that were associated with the exclusion of Cd and Hg. Overall, macroevolutionary conservation of heavy metal and stress response genes is important for improvement of forage crops by harnessing wild genetic variants in gene banks such as the Medicago HapMap collection.

Experimental and Field Data Support Range Expansion in an Allopolyploid Arabidopsis Owing to Parental Legacy of Heavy Metal Hyperaccumulation.

Empirical evidence is limited on whether allopolyploid species combine or merge parental adaptations to broaden habitats. The allopolyploid Arabidopsis kamchatica is a hybrid of the two diploid parents Arabidopsis halleri and Arabidopsis lyrata. A. halleri is a facultative heavy metal hyperaccumulator, and may be found in cadmium (Cd) and zinc (Zn) contaminated environments, as well as non-contaminated environments. A. lyrata is considered non-tolerant to these metals, but can be found in serpentine habitats. Therefore, the parents have adaptation to different environments. Here, we measured heavy metals in soils from native populations of A. kamchatica. We found that soil Zn concentration of nearly half of the sampled 40 sites was higher than the critical toxicity level. Many of the sites were near human construction, suggesting adaptation of A. kamchatica to artificially contaminated soils. Over half of the A. kamchatica populations had >1,000 µg g-1 Zn in leaf tissues. Using hydroponic treatments, most genotypes accumulated >3,000 µg g-1 Zn, with high variability among them, indicating substantial genetic variation in heavy metal accumulation. Genes involved in heavy metal hyperaccumulation showed an expression bias in the A. halleri-derived homeolog in widely distributed plant genotypes. We also found that two populations were found growing on serpentine soils. These data suggest that A. kamchatica can inhabit a range of both natural and artificial soil environments with high levels of ions that either of the parents specializes and that it can accumulate varying amount of heavy metals. Our field and experimental data provide a compelling example of combining genetic toolkits for soil adaptations to expand the habitat of an allopolyploid species.

Mercury-Tolerant Ensifer medicae Strains Display High Mercuric Reductase Activity and a Protective Effect on Nitrogen Fixation in Medicago truncatula Nodules Under Mercury Stress.

Mercury (Hg) is extremely toxic for all living organisms. Hg-tolerant symbiotic rhizobia have the potential to increase legume tolerance, and to our knowledge, the mechanisms underlying Hg tolerance in rhizobia have not been investigated to date. Rhizobial strains of Ensifer medicae, Rhizobium leguminosarum bv. trifolii and Bradyrhizobium canariense previously isolated from severely Hg-contaminated soils showed different levels of Hg tolerance. The ability of the strains to reduce mercury Hg2+ to Hg0, a volatile and less toxic form of mercury, was assessed using a Hg volatilization assay. In general, tolerant strains displayed high mercuric reductase activity, which appeared to be inducible in some strains when grown at a sub-lethal HgCl2 concentration. A strong correlation between Hg tolerance and mercuric reductase activity was observed for E. medicae strains, whereas this was not the case for the B. canariense strains, suggesting that additional Hg tolerance mechanisms could be playing a role in B. canariense. Transcript abundance from merA, the gene that encodes mercuric reductase, was quantified in tolerant and sensitive E. medicae and R. leguminosarum strains. Tolerant strains presented higher merA expression than sensitive ones, and an increase in transcript abundance was observed for some strains when bacteria were grown in the presence of a sub-lethal HgCl2 concentration. These results suggest a regulation of mercuric reductase in rhizobia. Expression of merA genes and mercuric reductase activity were confirmed in Medicago truncatula nodules formed by a sensitive or a tolerant E. medicae strain. Transcript accumulation in nodules formed by the tolerant strain increased when Hg stress was applied, while a significant decrease in expression occurred upon stress application in nodules formed by the Hg-sensitive strain. The effect of Hg stress on nitrogen fixation was evaluated, and in our experimental conditions, nitrogenase activity was not affected in nodules formed by the tolerant strain, while a significant decrease in activity was observed in nodules elicited by the Hg-sensitive bacteria. Our results suggest that the combination of tolerant legumes with tolerant rhizobia constitutes a potentially powerful tool in the bioremediation of Hg-contaminated soils.

Patterns of polymorphism and selection in the subgenomes of the allopolyploid Arabidopsis kamchatica.

Genome duplication is widespread in wild and crop plants. However, little is known about genome-wide selection in polyploids due to the complexity of duplicated genomes. In polyploids, the patterns of purifying selection and adaptive substitutions may be affected by masking owing to duplicated genes or homeologs as well as effective population size. Here, we resequence 25 accessions of the allotetraploid Arabidopsis kamchatica, which is derived from the diploid species A. halleri and A. lyrata. We observe a reduction in purifying selection compared with the parental species. Interestingly, proportions of adaptive non-synonymous substitutions are significantly positive in contrast to most plant species. A recurrent pattern observed in both frequency and divergence-diversity neutrality tests is that the genome-wide distributions of both subgenomes are similar, but the correlation between homeologous pairs is low. This may increase the opportunity of different evolutionary trajectories such as in the HMA4 gene involved in heavy metal hyperaccumulation.

Genome assembly and annotation of Arabidopsis halleri, a model for heavy metal hyperaccumulation and evolutionary ecology.

The self-incompatible species Arabidopsis halleri is a close relative of the self-compatible model plant Arabidopsis thaliana. The broad European and Asian distribution and heavy metal hyperaccumulation ability make A. halleri a useful model for ecological genomics studies. We used long-insert mate-pair libraries to improve the genome assembly of the A. halleri ssp. gemmifera Tada mine genotype (W302) collected from a site with high contamination by heavy metals in Japan. After five rounds of forced selfing, heterozygosity was reduced to 0.04%, which facilitated subsequent genome assembly. Our assembly now covers 196 Mb or 78% of the estimated genome size and achieved scaffold N50 length of 712 kb. To validate assembly and annotation, we used synteny of A. halleri Tada mine with a previously published high-quality reference assembly of a closely related species, Arabidopsis lyrata. Further validation of the assembly quality comes from synteny and phylogenetic analysis of the HEAVY METAL ATPASE4 (HMA4) and METAL TOLERANCE PROTEIN1 (MTP1) regions using published sequences from European A. halleri for comparison. Three tandemly duplicated copies of HMA4, key gene involved in cadmium and zinc hyperaccumulation, were assembled on a single scaffold. The assembly will enhance the genomewide studies of A. halleri as well as the allopolyploid Arabidopsis kamchatica derived from A. lyrata and A. halleri.

Conserved but Attenuated Parental Gene Expression in Allopolyploids: Constitutive Zinc Hyperaccumulation in the Allotetraploid Arabidopsis kamchatica.

Allopolyploidization combines parental genomes and often confers broader species distribution. However, little is known about parentally transmitted gene expression underlying quantitative traits following allopolyploidization because of the complexity of polyploid genomes. The allopolyploid species Arabidopsis kamchatica is a natural hybrid of the zinc hyperaccumulator Arabidopsis halleri and of the nonaccumulator Arabidopsis lyrata We found that A. kamchatica retained the ability to hyperaccumulate zinc from A. halleri and grows in soils with both low and high metal content. Hyperaccumulation of zinc by A. kamchatica was reduced to about half of A. halleri, but is 10-fold greater than A. lyrata Homeologs derived from A. halleri had significantly higher levels of expression of genes such as HEAVY METAL ATPASE4 (HMA4), METAL TRANSPORTER PROTEIN1 and other metal ion transporters than those derived from A. lyrata, which suggests cis-regulatory differences. A. kamchatica has on average about half the expression of these genes compared with A. halleri due to fixed heterozygosity inherent in allopolyploids. Zinc treatment significantly changed the ratios of expression of 1% of homeologous pairs, including genes putatively involved in metal homeostasis. Resequencing data showed a significant reduction in genetic diversity over a large genomic region (290 kb) surrounding the HMA4 locus derived from the A. halleri parent compared with the syntenic A. lyrata-derived region, which suggests different evolutionary histories. We also estimated that three A. halleri-derived HMA4 copies are present in A. kamchatica Our findings support a transcriptomic model in which environment-related transcriptional patterns of both parents are conserved but attenuated in the allopolyploids.

Gene duplication and genetic exchange drive the evolution of S-RNase-based self-incompatibility in Petunia.

Self-incompatibility (SI) systems in flowering plants distinguish self- and non-self pollen to prevent inbreeding. While other SI systems rely on the self-recognition between specific male- and female-determinants, the Solanaceae family has a non-self recognition system resulting in the detoxification of female-determinants of S-ribonucleases (S-RNases), expressed in pistils, by multiple male-determinants of S-locus F-box proteins (SLFs), expressed in pollen. It is not known how many SLF components of this non-self recognition system there are in Solanaceae species, or how they evolved. We identified 16-20 SLFs in each S-haplotype in SI Petunia, from a total of 168 SLF sequences using large-scale next-generation sequencing and genomic polymerase chain reaction (PCR) techniques. We predicted the target S-RNases of SLFs by assuming that a particular S-allele must not have a conserved SLF that recognizes its own S-RNase, and validated these predictions by transformation experiments. A simple mathematical model confirmed that 16-20 SLF sequences would be adequate to recognize the vast majority of target S-RNases. We found evidence of gene conversion events, which we suggest are essential to the constitution of a non-self recognition system and also contribute to self-compatible mutations.

The wheat powdery mildew genome shows the unique evolution of an obligate biotroph.

Wheat powdery mildew, Blumeria graminis forma specialis tritici, is a devastating fungal pathogen with a poorly understood evolutionary history. Here we report the draft genome sequence of wheat powdery mildew, the resequencing of three additional isolates from different geographic regions and comparative analyses with the barley powdery mildew genome. Our comparative genomic analyses identified 602 candidate effector genes, with many showing evidence of positive selection. We characterize patterns of genetic diversity and suggest that mildew genomes are mosaics of ancient haplogroups that existed before wheat domestication. The patterns of diversity in modern isolates suggest that there was no pronounced loss of genetic diversity upon formation of the new host bread wheat 10,000 years ago. We conclude that the ready adaptation of B. graminis f.sp. tritici to the new host species was based on a diverse haplotype pool that provided great genetic potential for pathogen variation.

Selection, genome-wide fitness effects and evolutionary rates in the model legume Medicago truncatula.

Sequence data for >20 000 annotated genes from 56 accessions of Medicago truncatula were used to identify potential targets of positive selection, the determinants of evolutionary rate variation and the relative importance of positive and purifying selection in shaping nucleotide diversity. Based upon patterns of intraspecific diversity and interspecific divergence, c. 50-75% of nonsynonymous polymorphisms are subject to strong purifying selection and 1% of the sampled genes harbour a signature of positive selection. Combining polymorphism with expression data, we estimated the distribution of fitness effects and found that the proportion of deleterious mutations is significantly greater for expressed genes than for genes with undetected transcripts (nonexpressed) in a previous RNA-seq experiment and greater for broadly expressed genes than those expressed in only a single tissue. Expression level is the strongest correlate of evolutionary rates at nonsynonymous sites, and despite multiple genomic features being significantly correlated with evolutionary rates, they explain less than 20% of the variation in nonsynonymous rates (dN) and <15% of the variation in either synonymous rates (dS) or dN:dS. Among putative targets of selection were genes involved in defence against pathogens and herbivores, genes with roles in mediating the relationship with rhizobial symbionts and one-third of annotated histone-lysine methyltransferases. Adaptive evolution of the methyltransferases suggests that positive selection in gene expression may have occurred through evolution of enzymes involved in epigenetic modification.

Candidate genes and genetic architecture of symbiotic and agronomic traits revealed by whole-genome, sequence-based association genetics in Medicago truncatula.

Genome-wide association study (GWAS) has revolutionized the search for the genetic basis of complex traits. To date, GWAS have generally relied on relatively sparse sampling of nucleotide diversity, which is likely to bias results by preferentially sampling high-frequency SNPs not in complete linkage disequilibrium (LD) with causative SNPs. To avoid these limitations we conducted GWAS with >6 million SNPs identified by sequencing the genomes of 226 accessions of the model legume Medicago truncatula. We used these data to identify candidate genes and the genetic architecture underlying phenotypic variation in plant height, trichome density, flowering time, and nodulation. The characteristics of candidate SNPs differed among traits, with candidates for flowering time and trichome density in distinct clusters of high linkage disequilibrium (LD) and the minor allele frequencies (MAF) of candidates underlying variation in flowering time and height significantly greater than MAF of candidates underlying variation in other traits. Candidate SNPs tagged several characterized genes including nodulation related genes SERK2, MtnodGRP3, MtMMPL1, NFP, CaML3, MtnodGRP3A and flowering time gene MtFD as well as uncharacterized genes that become candidates for further molecular characterization. By comparing sequence-based candidates to candidates identified by in silico 250K SNP arrays, we provide an empirical example of how reliance on even high-density reduced representation genomic makers can bias GWAS results. Depending on the trait, only 30-70% of the top 20 in silico array candidates were within 1 kb of sequence-based candidates. Moreover, the sequence-based candidates tagged by array candidates were heavily biased towards common variants; these comparisons underscore the need for caution when interpreting results from GWAS conducted with sparsely covered genomes.

Phylogenetic signal variation in the genomes of Medicago (Fabaceae).

Genome-scale data offer the opportunity to clarify phylogenetic relationships that are difficult to resolve with few loci, but they can also identify genomic regions with evolutionary history distinct from that of the species history. We collected whole-genome sequence data from 29 taxa in the legume genus Medicago, then aligned these sequences to the Medicago truncatula reference genome to confidently identify 87 596 variable homologous sites. We used this data set to estimate phylogenetic relationships among Medicago species, to investigate the number of sites needed to provide robust phylogenetic estimates and to identify specific genomic regions supporting topologies in conflict with the genome-wide phylogeny. Our full genomic data set resolves relationships within the genus that were previously intractable. Subsampling the data reveals considerable variation in phylogenetic signal and power in smaller subsets of the data. Even when sampling 5000 sites, no random sample of the data supports a topology identical to that of the genome-wide phylogeny. Phylogenetic relationships estimated from 500-site sliding windows revealed genome regions supporting several alternative species relationships among recently diverged taxa, consistent with the expected effects of deep coalescence or introgression in the recent history of Medicago.

Fitness costs of butterfly oviposition on a lethal non-native plant in a mixed native and non-native plant community.

Non-native plants may be unpalatable or toxic, but have oviposition cues similar to native plants used by insects. The herbivore will then oviposit on the plant, but the offspring will be unable to develop. While such instances have been described previously, the fitness costs at the population level in the wild due to the presence of the lethal host have not been quantified, for this or other related systems. We quantified the fitness cost in the field for the native butterfly Pieris macdunnoughii in the presence of the non-native crucifer Thlaspi arvense, based on the spatial distributions of host plants, female butterflies and eggs in the habitat and the survival of the larvae in the wild. We found that 2.9% of eggs were laid on T. arvense on average, with a survival probability of 0, yielding a calculated fitness cost of 3.0% (95% confidence interval 1.7-3.6%) due to the presence of the non-native in the plant community. Survival probability to the pre-pupal stage for eggs laid on two native crucifers averaged 1.6% over 2 years. The magnitude of the fitness cost will vary temporally and spatially as a function of the relative abundance of the non-native plant. We propose that the fine-scale spatial structure of the plant community relative to the butterflies' dispersal ability, combined with the females' broad habitat use, contributes to the fitness costs associated with the non-native plant and the resulting evolutionary trap.

Fine-scale population recombination rates, hotspots, and correlates of recombination in the Medicago truncatula genome.

Recombination rates vary across the genome and in many species show significant relationships with several genomic features, including distance to the centromere, gene density, and GC content. Studies of fine-scale recombination rates have also revealed that in several species, there are recombination hotspots, that is, short regions with recombination rates 10-100 greater than those in surrounding regions. In this study, we analyzed whole-genome resequence data from 26 accessions of the model legume Medicago truncatula to gain insight into the genomic features that are related to high- and low-recombination rates and recombination hotspots at 1 kb scales. We found that high-recombination regions (1-kb windows among those in the highest 5% of the distribution) on all three chromosomes were significantly closer to the centromere, had higher gene density, and lower GC content than low-recombination windows. High-recombination windows are also significantly overrepresented among some gene functional categories-most strongly NB-ARC and LRR genes, both of which are important in plant defense against pathogens. Similar to high-recombination windows, recombination hotspots (1-kb windows with significantly higher recombination than the surrounding region) are significantly nearer to the centromere than nonhotspot windows. By contrast, we detected no difference in gene density or GC content between hotspot and nonhotspot windows. Using linear model wavelet analysis to examine the relationship between recombination and genomic features across multiple spatial scales, we find a significant negative correlation with distance to the centromere across scales up to 512 kb, whereas gene density and GC content show significantly positive and negative correlations, respectively, only up to 64 kb. Correlations between recombination and genomic features, particularly gene density and polymorphism, suggest that they are scale dependent and need to be assessed at scales relevant to the evolution of those features.

Evolutionary genetics of an S-like polymorphism in Papaveraceae with putative function in self-incompatibility.

BACKGROUND: Papaver rhoeas possesses a gametophytic self-incompatibility (SI) system not homologous to any other SI mechanism characterized at the molecular level. Four previously published full length stigmatic S-alleles from the genus Papaver exhibited remarkable sequence divergence, but these studies failed to amplify additional S-alleles despite crossing evidence for more than 60 S-alleles in Papaver rhoeas alone. METHODOLOGY/PRINCIPAL FINDINGS: Using RT-PCR we identified 87 unique putative stigmatic S-allele sequences from the Papaveraceae Argemone munita, Papaver mcconnellii, P. nudicuale, Platystemon californicus and Romneya coulteri. Hand pollinations among two full-sib families of both A. munita and P. californicus indicate a strong correlation between the putative S-genotype and observed incompatibility phenotype. However, we also found more than two S-like sequences in some individuals of A. munita and P. californicus, with two products co-segregating in both full-sib families of P. californicus. Pairwise sequence divergence estimates within and among taxa show Papaver stigmatic S-alleles to be the most variable with lower divergence among putative S-alleles from other Papaveraceae. Genealogical analysis indicates little shared ancestral polymorphism among S-like sequences from different genera. Lack of shared ancestral polymorphism could be due to long divergence times among genera studied, reduced levels of balancing selection if some or all S-like sequences do not function in incompatibility, population bottlenecks, or different levels of recombination among taxa. Preliminary estimates of positive selection find many sites under selective constraint with a few undergoing positive selection, suggesting that self-recognition may depend on amino acid substitutions at only a few sites. CONCLUSIONS/SIGNIFICANCE: Because of the strong correlation between genotype and SI phenotype, sequences reported here represent either functional stylar S-alleles, tightly linked paralogs of the S-locus or a combination of both. The considerable complexity revealed in this study shows we have much to learn about the evolutionary dynamics of self-incompatibility systems.

Whole-genome nucleotide diversity, recombination, and linkage disequilibrium in the model legume Medicago truncatula.

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.

Differential strengths of selection on S-RNases from Physalis and Solanum (Solanaceae).

BACKGROUND: The S-RNases of the Solanaceae are highly polymorphic self-incompatibility (S-) alleles subject to strong balancing selection. Relatively recent diversification of S-alleles has occurred in the genus Physalis following a historical restriction of S-allele diversity. In contrast, the genus Solanum did not undergo a restriction of S-locus diversity and its S-alleles are generally much older. Because recovery from reduced S-locus diversity should involve increased selection, we employ a statistical framework to ask whether S-locus selection intensities are higher in Physalis than Solanum. Because different S-RNase lineages diversify in Physalis and Solanum, we also ask whether different sites are under selection in different lineages. RESULTS: Maximum-likelihood and Bayesian coalescent methods found higher intensities of selection and more sites under significant positive selection in the 48 Physalis S-RNase alleles than the 49 from Solanum. Highest posterior densities of dN/dS (ω) estimates show that the strength of selection is greater for Physalis at 36 codons. A nested maximum likelihood method was more conservative, but still found 16 sites with greater selection in Physalis. Neither method found any codons under significantly greater selection in Solanum. A random effects likelihood method that examines data from both taxa jointly confirmed higher selection intensities in Physalis, but did not find different proportions of sites under selection in the two datasets. The greatest differences in strengths of selection were found in the most variable regions of the S-RNases, as expected if these regions encode self-recognition specificities. Clade-specific likelihood models indicated some codons were under greater selection in background Solanum lineages than in specific lineages of Physalis implying that selection on sites may differ among lineages. CONCLUSIONS: Likelihood and Bayesian methods provide a statistical approach to testing differential selection across populations or species. These tests appear robust to the levels of polymorphism found in diverse S-allele collections subject to strong balancing selection. As predicted, the intensity of selection at the S-locus was higher in the taxon with more recent S-locus diversification. This is the first confirmation by statistical test of differing selection intensities among self-incompatibility alleles from different populations or species.