Lex E. Flagel

Homoeolog expression bias and expression level dominance in allopolyploids

Polyploidy is now recognized as a characteristic feature of all angiosperm genomes (Jiao et al., 2011), and remains an important speciation process today (Wendel, 2000; Comai, 2005; Doyle et al., 2008; Leitch & Leitch, 2008; Soltis & Soltis, 2009; Soltis et al., 2010). In allopolyploids, genomic merger and doubling are associated with myriad non-Mendelian interactions and processes, including sequence elimination (Shaked et al., 2001; Ozkan et al., 2003; Han et al., 2005; Skalicka et al., 2005; Anssour et al., 2009; Tate et al., 2009; Jackson & Chen, 2010), alterations of epigenetic marks (Shaked et al., 2001;Madlung et al., 2002; Rapp&Wendel, 2005; Chen, 2007; Doyle et al., 2008; Kovarik et al., 2008b; Soltis & Soltis, 2009; Soltis et al., 2010), activation of genes and retroelements (O’Neill et al., 1998; Kashkush et al., 2003; Kraitshtein et al., 2010) and several kinds of homoeologous interactions and exchanges (Gaeta et al., 2007; Kovarik et al., 2008a; Salmon et al., 2010; Szadkowski et al., 2010). Changes in duplicate gene expression are no less diverse, spanning the spectrum from expression conservation, relative to that of the diploid progenitors, to silencing of one homoeolog, to novel patterns of upand down-regulation (transgressive expression). Each of these transcriptomic responses varies in magnitude among allopolyploid species and individuals, among tissues and organ types within any one system, and with respect to the time since polyploid formation (Flagel et al., 2008; Flagel & Wendel, 2010). The phenotypic consequences of alterations in gene expression associated with hybridization and polyploidy are many and varied (Ni et al., 2009; Swanson-Wagner et al., 2009), underscoring the importance of understanding the expression level consequences of genomemerger and doubling. The advent and subsequent widespread utilization ofmicroarray and next-generation sequencing technologies has led to a rapid increase in explorations of gene expression in a variety of polyploid plants. These many efforts have generated a sufficient body of empirical data that generalizations are beginning to emerge concerning transcriptome changes in allopolyploids. For example, in every allopolyploid examined to date, some fraction of the duplicate gene pairs will be expressed unequally, and this suite of unequally expressed genes may itself favor one of the co-resident genomes, leading to a transcriptome that is unequally expressed with respect to the component genomes. While these generalizations are broadly applicable, much remains to be learned regarding the mechanistic underpinnings of duplicate gene expression change, the proximate and ultimate causes of inter-taxon and inter-organ variation in the response dynamics to polyploidy, and the functional, ecological, and evolutionary significance of duplicate gene expression modification. In addition to unequal expression of two homoeologs, other phenomena have been described which are even more poorly understood and for which fewer examples have yet been published. One of these is the concept of genome dominance (or genome expression dominance), which describes the expression condition in an allopolyploid where, for a given gene, the total expression of homoeologs is statistically the same as only one of the polyploid parents. This phenomenon was originally described for cotton allopolyploids by Rapp et al. (2009), confirmed and extended by Flagel & Wendel (2010), and subsequently described for both Spartina (Chelaifa et al., 2010) and Coffea (Bardil et al., 2011). This phenomenon is distinct from homoeolog expression bias (sometimes referred to as transcriptome dominance on a genome-wide basis), which describes the relative expression of homoeologs. Moreover, similar words are being used for rather different phenomena. Schnable et al. (2011), for example, invoked the term genomic dominance in maize, in a paper in which they demonstrated that the two subgenomes derived from the most recent polyploidy event in maize have experienced differential gene loss, with an accompanying gene expression bias favoring the more conserved subgenome (Schnable et al., 2011). By other accounts (Chen, 2007; Flagel & Wendel, 2010), this would be considered homoeolog expression bias (or transcriptome dominance) of ancient homoeologs. This inconsistency of conceptual application of the term genomic dominance also applies to the preferential expression of one subgenome of wheat (Akhunova et al., 2010), and to the patterns of biased expression in the fractionated subgenomes of paleohexaploid Brassica rapa (Cheng et al., 2012; Tang et al., 2012). This semantic and conceptual confusion appears to be gaining foothold in the literature; the phenomenon of preferential expression of one parental genome relative to the other in a polyploid species is termed genomic dominance in two recent reviews (Freeling et al., 2012; Schnable et al., 2012), citing both Schnable et al. (2011) and Flagel & Wendel (2010), and the term has also been applied to genomic modifications (Nicolas et al., 2012). Further complicating matters is the classical genetic concept associated with the term ‘dominance’, which conveys the relative expression hierarchy among a set of alleles. Against this backdrop of terminological and conceptual inconsistency, we thought it might be useful to briefly review the primary phenotypes of gene expression modification associated with allopolyploidy. Toward that end we describe and distinguish expression pattern changes observed in hybrid and polyploid species, and suggest a terminology (homoeolog expression bias and expression level dominance; Table 1; Fig. 1) that we hope will increase clarity of communication.

Duplicate gene evolution, homoeologous recombination, and transcriptome characterization in allopolyploid cotton

BackgroundModern allotetraploid cotton contains an “A” and “D” genome from an ancestral polyploidy event that occurred approximately 1–2 million years ago. Diploid A- and D-genome species can be compared to the A- and D-genomes found within these allotetraploids to make evolutionary inferences about polyploidy. In this paper we present a comprehensive EST assembly derived from diploid and model allotetraploid cottons and demonstrate several evolutionary inferences regarding genic evolution that can be drawn from these data.ResultsWe generated a set of cotton expressed sequence tags (ESTs), comprising approximately 4.4 million Sanger and next-generation (454) transcripts supplemented by approximately 152 million Illumina reads from diploid and allotetraploid cottons. From the EST alignments we inferred 259,192 genome-specific single nucleotide polymorphisms (SNPs). Molecular evolutionary analyses of protein-coding regions demonstrate that the rate of nucleotide substitution has increased among both allotetraploid genomes relative to the diploids, and that the ratio of nonsynonymous to synonymous substitutions has increased in one of the two polyploid lineages we sampled. We also use these SNPs to show that a surprisingly high percentage of duplicate genes (~7 %) show a signature of non-independent evolution in the allotetraploid nucleus, having experienced one or more episodes of nonreciprocal homoeologous recombination (NRHR).ConclusionsIn this study we characterize the functional and mutational properties of the cotton transcriptome, produce a large genome-specific SNP database, and detect illegitimate genetic exchanges between duplicate genomes sharing a common allotetraploid nucleus. Our findings have important implications for our understanding of the consequences of polyploidy and duplicate gene evolution. We demonstrate that cotton genes have experienced an increased rate of molecular evolution following duplication by polyploidy, and that polyploidy has enabled considerable levels of nonreciprocal exchange between homoeologous genes.

Speciation and introgression between Mimulus nasutus and Mimulus guttatus.

Mimulus guttatus and M. nasutus are an evolutionary and ecological model sister species pair differentiated by ecology, mating system, and partial reproductive isolation. Despite extensive research on this system, the history of divergence and differentiation in this sister pair is unclear. We present and analyze a population genomic data set which shows that M. nasutus budded from a central Californian M. guttatus population within the last 200 to 500 thousand years. In this time, the M. nasutus genome has accrued genomic signatures of the transition to predominant selfing, including an elevated proportion of nonsynonymous variants, an accumulation of premature stop codons, and extended levels of linkage disequilibrium. Despite clear biological differentiation, we document genomic signatures of ongoing, bidirectional introgression. We observe a negative relationship between the recombination rate and divergence between M. nasutus and sympatric M. guttatus samples, suggesting that selection acts against M. nasutus ancestry in M. guttatus.

Parallel Domestication, Convergent Evolution and Duplicated Gene Recruitment in Allopolyploid Cotton

A putative advantage of allopolyploidy is the possibility of differential selection of duplicated (homeologous) genes originating from two different progenitor genomes. In this note we explore this hypothesis using a high throughput, SNP-specific microarray technology applied to seed trichomes (cotton) harvested from three developmental time points in wild and modern accessions of two independently domesticated cotton species, Gossypium hirsutum and G. barbadense. We show that homeolog expression ratios are dynamic both developmentally and over the several-thousand-year period encompassed by domestication and crop improvement, and that domestication increased the modulation of homeologous gene expression. In both species, D-genome expression was preferentially enhanced under human selection pressure, but for nonoverlapping sets of genes for the two independent domestication events. Our data suggest that human selection may have operated on different components of the fiber developmental genetic program in G. hirsutum and G. barbadense, leading to convergent rather than parallel genetic alterations and resulting morphology.

Parallel up-regulation of the profilin gene family following independent domestication of diploid and allopolyploid cotton (Gossypium)

Cotton is remarkable among our major crops in that four species were independently domesticated, two allopolyploids and two diploids. In each case thousands of years of human selection transformed sparsely flowering, perennial shrubs into highly productive crops with seeds bearing the vastly elongated and abundant single-celled hairs that comprise modern cotton fiber. The genetic underpinnings of these transformations are largely unknown, but comparative gene expression profiling experiments have demonstrated up-regulation of profilin accompanying domestication in all three species for which wild forms are known. Profilins are actin monomer binding proteins that are important in cytoskeletal dynamics and in cotton fiber elongation. We show that Gossypium diploids contain six profilin genes (GPRF1–GPRF6), located on four different chromosomes (eight chromosomes in the allopolyploid). All but one profilin (GPRF6) are expressed during cotton fiber development, and both homeologs of GPRF1–GPRF5 are expressed in fibers of the allopolyploids. Remarkably, quantitative RT-PCR and RNAseq data demonstrate that GPRF1–GPRF5 are all up-regulated, in parallel, in the three independently domesticated cottons in comparison with their wild counterparts. This result was additionally supported by iTRAQ proteomic data. In the allopolyploids, there This usage of novel should be fine, since it refers to a novel evolutionary process, not a novel discovery has been novel recruitment of the sixth profilin gene (GPRF6) as a result of domestication. This parallel up-regulation of an entire gene family in multiple species in response to strong directional selection is without precedent and suggests unwitting selection on one or more upstream transcription factors or other proteins that coordinately exercise control over profilin expression.

Jeans, Genes, and Genomes: Cotton as a Model for Studying Polyploidy

We present an overview of the cotton genus (Gossypium) as a model for the study of polyploidy. A synopsis of the origin and evolution of polyploid cotton is provided, offering an organismal framework and phylogenetic perspective that is critical for understanding modes and mechanisms of gene and genome evolution. Sequence data from thousands of genes implicate a mid-Pleistocene (1–2 mya) origin of polyploid cotton, following trans-oceanic dispersal of an Old World, A-genome diploid to the New World and subsequent hybridization with an indigenous D-genome diploid. This chance biological reunion, occurring after 5–10 million years of diploid evolution in isolation, has led to an array of molecular genetic interactions in the newly formed allopolyploid lineage, including nonreciprocal homoeologous recombination and perhaps other forms of interlocus concerted evolution, differential rates of genomic evolution, intergenomic spread of transposable elements, and myriad forms of alterations in duplicate expression relative to that experienced in the ancestral diploids. The latter include developmental, organ-, tissue-, and cell-specific biases in homoeologous gene expression, which can be sensitive to various forms of environmental perturbation and stress. The allopolyploid Gossypium transcriptome is exceptionally dynamic, with homoeolog expression ratios being subject to change even during development of the single-celled cotton fiber. Expression evolution is temporally partitioned into changes accompanying genome merger (hybridization) at the diploid level, polyploidization, and longer term evolution at the allopolyploid level. Evidence indicates that allopolyploidy facilitated colonization of a new ecological niche for the genus and led to an enhanced capacity for developing agronomically superior cotton varieties. The myriad mechanisms that underlie genomic and regulatory evolution are suggested to have contributed to both ecological success and agronomic potential.

Phylogenetic, morphological, and chemotaxonomic incongruence in the North American endemic genus Echinacea

The study of recently formed species is important because it can help us to better understand organismal divergence and the speciation process. However, these species often present difficult challenges in the field of molecular phylogenetics because the processes that drive molecular divergence can lag behind phenotypic divergence. In the current study we show that species of the recently diverged North American endemic genus of purple coneflower, Echinacea, have low levels of molecular divergence. Data from three nuclear loci and two plastid loci provide neither resolved topologies nor congruent hypotheses about species-level relationships. This lack of phylogenetic resolution is likely due to the combined effects of incomplete lineage sorting, hybridization, and backcrossing following secondary contact. The poor resolution provided by molecular markers contrasts previous studies that found well-resolved and taxonomically supported relationships from metabolic and morphological data. These results suggest that phenotypic canalization, resulting in identifiable morphological species, has occurred rapidly within Echinacea. Conversely, molecular signals have been distorted by gene flow and incomplete lineage sorting. Here we explore the impact of natural history on the genetic organization and phylogenetic relationships of Echinacea.

Rapid molecular evolution across amniotes of the IIS/TOR network

Significance Comparative analyses of central molecular networks uncover variation that can be targeted by biomedical research to develop insights and interventions into disease. The insulin/insulin-like signaling and target of rapamycin (IIS/TOR) molecular network regulates metabolism, growth, and aging. With the development of new molecular resources for reptiles, we show that genes in IIS/TOR are rapidly evolving within amniotes (mammals and reptiles, including birds). Additionally, we find evidence of natural selection that diversified the hormone-receptor binding relationships that initiate IIS/TOR signaling. Our results uncover substantial variation in the IIS/TOR network within and among amniotes and provide a critical step to unlocking information on vertebrate patterns of genetic regulation of metabolism, modes of reproduction, and rates of aging. The insulin/insulin-like signaling and target of rapamycin (IIS/TOR) network regulates lifespan and reproduction, as well as metabolic diseases, cancer, and aging. Despite its vital role in health, comparative analyses of IIS/TOR have been limited to invertebrates and mammals. We conducted an extensive evolutionary analysis of the IIS/TOR network across 66 amniotes with 18 newly generated transcriptomes from nonavian reptiles and additional available genomes/transcriptomes. We uncovered rapid and extensive molecular evolution between reptiles (including birds) and mammals: (i) the IIS/TOR network, including the critical nodes insulin receptor substrate (IRS) and phosphatidylinositol 3-kinase (PI3K), exhibit divergent evolutionary rates between reptiles and mammals; (ii) compared with a proxy for the rest of the genome, genes of the IIS/TOR extracellular network exhibit exceptionally fast evolutionary rates; and (iii) signatures of positive selection and coevolution of the extracellular network suggest reptile- and mammal-specific interactions between members of the network. In reptiles, positively selected sites cluster on the binding surfaces of insulin-like growth factor 1 (IGF1), IGF1 receptor (IGF1R), and insulin receptor (INSR); whereas in mammals, positively selected sites clustered on the IGF2 binding surface, suggesting that these hormone-receptor binding affinities are targets of positive selection. Further, contrary to reports that IGF2R binds IGF2 only in marsupial and placental mammals, we found positively selected sites clustered on the hormone binding surface of reptile IGF2R that suggest that IGF2R binds to IGF hormones in diverse taxa and may have evolved in reptiles. These data suggest that key IIS/TOR paralogs have sub- or neofunctionalized between mammals and reptiles and that this network may underlie fundamental life history and physiological differences between these amniote sister clades.

The standing pool of genomic structural variation in a natural population of Mimulus guttatus.

Major unresolved questions in evolutionary genetics include determining the contributions of different mutational sources to the total pool of genetic variation in a species, and understanding how these different forms of genetic variation interact with natural selection. Recent work has shown that structural variants (SVs) (insertions, deletions, inversions, and transpositions) are a major source of genetic variation, often outnumbering single nucleotide variants in terms of total bases affected. Despite the near ubiquity of SVs, major questions about their interaction with natural selection remain. For example, how does the allele frequency spectrum of SVs differ when compared with single nucleotide variants? How often do SVs affect genes, and what are the consequences? To begin to address these questions, we have systematically identified and characterized a large set of submicroscopic insertion and deletion (indel) variants (between 1 and 200 kb in length) among ten inbred lines from a single natural population of the plant species Mimulus guttatus. After extensive computational filtering, we focused on a set of 4,142 high-confidence indels that showed an experimental validation rate of 73%. All but one of these indels were less than 200 kb. Although the largest were generally at lower frequencies in the population, a surprising number of large indels are at intermediate frequencies. Although indels overlapping with genes were much rarer than expected by chance, approximately 600 genes were affected by an indel. Nucleotide-binding site leucine-rich repeat (NBS–LRR) defense response genes were the most enriched among the gene families affected. Most indels associated with genes were rare and appeared to be under purifying selection, though we do find four high-frequency derived insertion alleles that show signatures of recent positive selection.

Evidence of Natural Selection Acting on a Polymorphic Hybrid Incompatibility Locus in Mimulus

As a common cause of reproductive isolation in diverse taxa, hybrid incompatibilities are fundamentally important to speciation. A key question is which evolutionary forces drive the initial substitutions within species that lead to hybrid dysfunction. Previously, we discovered a simple genetic incompatibility that causes nearly complete male sterility and partial female sterility in hybrids between the two closely related yellow monkeyflower species Mimulus guttatus and M. nasutus. In this report, we fine map the two major incompatibility loci—hybrid male sterility 1 (hms1) and hybrid male sterility 2 (hms2)—to small nuclear genomic regions (each <70 kb) that include strong candidate genes. With this improved genetic resolution, we also investigate the evolutionary dynamics of hms1 in a natural population of M. guttatus known to be polymorphic at this locus. Using classical genetic crosses and population genomics, we show that a 320-kb region containing the hms1 incompatibility allele has risen to intermediate frequency in this population by strong natural selection. This finding provides direct evidence that natural selection within plant species can lead to hybrid dysfunction between species.