Joseph P. Gallagher

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.

Ancient Gene Duplicates in Gossypium (Cotton) Exhibit Near-Complete Expression Divergence

Whole genome duplication (WGD) is widespread in flowering plants and is a driving force in angiosperm diversification. The redundancy introduced by WGD allows the evolution of novel gene interactions and functions, although the patterns and processes of diversification are poorly understood. We identified ∼2,000 pairs of paralogous genes in Gossypium raimondii (cotton) resulting from an approximately 60 My old 5- to 6-fold ploidy increase. Gene expression analyses revealed that, in G. raimondii, 99.4% of the gene pairs exhibit differential expression in at least one of the three tissues (petal, leaf, and seed), with 93% to 94% exhibiting differential expression on a per-tissue basis. For 1,666 (85%) pairs, differential expression was observed in all tissues. These observations were mirrored in a time series of G. raimondii seed, and separately in leaf, petal, and seed of G. arboreum, indicating expression level diversification before species divergence. A generalized linear model revealed 92.4% of the paralog pairs exhibited expression divergence, with most exhibiting significant gene and tissue interactions indicating complementary expression patterns in different tissues. These data indicate massive, near-complete expression level neo- and/or subfunctionalization among ancient gene duplicates, suggesting these processes are essential in their maintenance over ∼60 Ma.

Persistence of Subgenomes in Paleopolyploid Cotton after 60 My of Evolution

The importance of whole-genome multiplication (WGM) in plant evolution has long been recognized. In flowering plants, WGM is both ubiquitous and in many lineages cyclical, each round followed by substantial gene loss (fractionation). This process may be biased with respect to duplicated chromosomes, often with overexpression of genes in less fractionated relative to more fractionated regions. This bias is hypothesized to arise through downregulation of gene expression through silencing of local transposable elements (TEs). We assess differences in gene expression between duplicated regions of the paleopolyploid cotton genome and demonstrate that the rate of fractionation is negatively correlated with gene expression. We examine recent hypotheses regarding the source of fractionation bias and show that TE-mediated, positional downregulation is absent in the modern cotton genome, seemingly excluding this phenomenon as the primary driver of biased gene loss. Nevertheless, the paleo subgenomes of diploid cotton are still distinguishable with respect to TE content, targeting of 24-nt-small interfering RNAs and GC content, despite approximately 60 My of evolution. We propose that repeat content per se and differential recombination rates may drive biased fractionation following WGM. These data highlight the likely importance of ancient genomic fractionation biases in shaping modern crop genomes.

Re-evaluating the phylogeny of allopolyploid Gossypium L.

The formation of allopolyploid cotton precipitated a rapid diversification and colonization of dry coastal American tropical and subtropical regions. Previous phylogenetic analyses, combined with molecular divergence analyses, have offered a temporal framework for this radiation, but provide only weak support for some of the resolved branches. Moreover, these earlier analyses did not include the recently recognized sixth polyploid species, G. ekmanianum Wittmack. Here we use targeted sequence capture of multiple loci in conjunction with both concatenated and Bayesian concordance analyses to reevaluate the phylogeny of allopolyploid cotton species. Although phylogenetic resolution afforded by individual genes is often low, sufficient signal was attained both through the concatenated and concordance analyses to provide robust support for the Gossypium polyploid clade, which is reported here.

Molecular confirmation of species status for the allopolyploid cotton species, Gossypium ekmanianum Wittmack

Understanding the relationship between domesticated crop species and their wild relatives is paramount to germplasm maintenance and the utilization of wild relatives in breeding programs. Recently, Gossypium ekmanianum was resurrected as an independent species based on morphological analysis of specimens obtained from the Dominican Republic, where the original type specimen was collected. The molecular data presented here support the recognition of G. ekmanianum Wittmack as a distinct species that is phylogenetically close to G. hirsutum L. Analyses of chloroplast DNA data reveal species-specific, indel polymorphisms that unambiguously distinguish G. ekmanianum samples from other polyploid congeners. Furthermore, analysis of accessions that originated from the Dominican Republic demonstrate the cryptic inclusion of this sister taxon within the US National Plant Germplasm System, a germplasm collection maintained for diversity preservation and future breeding resources. The data presented here indicate that “wild” G. hirsutum accessions may include the closely related G. ekmanianum, and provide a method to easily distinguish the two.

A New Species of Cotton from Wake Atoll, Gossypium stephensii (Malvaceae)

Abstract Wake Atoll is an isolated chain of three islets located in the Western Pacific. Included in its endemic flora is a representative of the genus Gossypium colloquially referred to as Wake Island cotton. Stanley G. Stephens pointed out that “Wake Island cotton does not resemble closely either the Caribbean or other Pacific forms.” Taking into consideration morphological distinctions, the geographic isolation of Wake Atoll, and newly generated molecular data presented here, we conclude that the cottons of Wake Atoll do in fact represent a new species of Gossypium, here named Gossypium stephensii. This name is chosen to commemorate the eminent natural historian, evolutionary geneticist, and cotton biologist, S. G. Stephens.

A Transcriptome Profile for Developing Seed of Polyploid Cotton

Cotton ranks among the worlds important oilseed crops, yet relative to other oilseeds there are few studies of oil‐related biosynthetic and regulatory pathways. We present global transcriptome analyses of cotton seed development using RNA‐seq and four developmental time‐points. Because Upland cotton (Gossypium hirsutum L.) is an allopolyploid containing two genomes (A/D), we partitioned expression into the individual contributions of each homeologous gene copy. Data were explored with respect to genic and subgenomic patterns of expression, globally and with respect to seed pathways and networks. The most dynamic period of transcriptome change is from 20–30 d postanthesis (DPA), with about 20% of genes showing homeolog expression bias. Co‐expression analysis shows largely congruent homeolog networks, but also homeolog‐specific divergence. Functional enrichment tests show that flavonoid biosynthesis and lipid related genes were significantly represented early and later in seed development, respectively. An involvement of new features in oil biosynthesis was found, like the contribution of DGAT3 (diacylglycerol acyltransferase) to the total triglyceride expression pool. Also, catechin‐based and epicatechin‐based proanthocyanidin expression are reciprocally biased with respect to homeolog usage. This study provides the first temporal analysis of duplicated gene expression in cotton seed and a resource for understanding new aspects of oil and flavonoid biosynthetic processes.

CenH3 Evolution in Diploids and Polyploids of Three Angiosperm Genera

BackgroundCentromeric DNA sequences alone are neither necessary nor sufficient for centromere specification. The centromere specific histone, CenH3, evolves rapidly in many species, perhaps as a coevolutionary response to rapidly evolving centromeric DNA. To gain insight into CenH3 evolution, we characterized patterns of nucleotide and protein diversity among diploids and allopolyploids within three diverse angiosperm genera, Brassica, Oryza, and Gossypium (cotton), with a focus on evidence for diversifying selection in the various domains of the CenH3 gene. In addition, we compare expression profiles and alternative splicing patterns for CenH3 in representatives of each genus.ResultsAll three genera retain both duplicated CenH3 copies, while Brassica and Gossypium exhibit pronounced homoeologous expression level bias. Comparisons among genera reveal shared and unique aspects of CenH3 evolution, variable levels of diversifying selection in different CenH3 domains, and that alternative splicing contributes significantly to CenH3 diversity.ConclusionsSince the N terminus is subject to diversifying selection but the DNA binding domains do not appear to be, rapidly evolving centromere sequences are unlikely to be the primary driver of CenH3 sequence diversification. At present, the functional explanation for the diversity generated by both conventional protein evolution in the N terminal domain, as well as alternative splicing, remains unexplained.

Insights into the Ecology and Evolution of Polyploid Plants through Network Analysis

Polyploidy is a widespread phenomenon throughout eukaryotes, with important ecological and evolutionary consequences. Although genes operate as components of complex pathways and networks, polyploid changes in genes and gene expression have typically been evaluated as either individual genes or as a part of broad‐scale analyses. Network analysis has been fruitful in associating genomic and other ‘omic’‐based changes with phenotype for many systems. In polyploid species, network analysis has the potential not only to facilitate a better understanding of the complex ‘omic’ underpinnings of phenotypic and ecological traits common to polyploidy, but also to provide novel insight into the interaction among duplicated genes and genomes. This adds perspective to the global patterns of expression (and other ‘omic’) change that accompany polyploidy and to the patterns of recruitment and/or loss of genes following polyploidization. While network analysis in polyploid species faces challenges common to other analyses of duplicated genomes, present technologies combined with thoughtful experimental design provide a powerful system to explore polyploid evolution. Here, we demonstrate the utility and potential of network analysis to questions pertaining to polyploidy with an example involving evolution of the transgressively superior cotton fibres found in polyploid Gossypium hirsutum. By combining network analysis with prior knowledge, we provide further insights into the role of profilins in fibre domestication and exemplify the potential for network analysis in polyploid species.

Candidate Gene Identification of Flowering Time Genes in Cotton

Flowering time control is critically important to all sexually reproducing angiosperms in both natural ecological and agronomic settings. Accordingly, there is much interest in defining the genes involved in the complex flowering‐time network and how these respond to natural and artificial selection, the latter often entailing transitions in day‐length responses. Here we describe a candidate gene analysis in the cotton genus Gossypium, which uses homologs from the well‐described Arabidopsis flowering network to bioinformatically and phylogenetically identify orthologs in the published genome sequence from G. raimondii Ulbr., one of the two model diploid progenitors of the commercially important allopolyploid cottons, G. hirsutum L. and G. barbadense L. Presence and patterns of expression were evaluated from 13 aboveground tissues related to flowering for each of the candidate genes using allopolyploid G. hirsutum as a model. Furthermore, we use a comparative context to determine copy number variability of each key gene family across 10 published angiosperm genomes. Data suggest a pattern of repeated loss of duplicates following ancient whole‐genome doubling events in diverse lineages. The data presented here provide a foundation for understanding both the parallel evolution of day‐length neutrality in domesticated cottons and the flowering‐time network, in general, in this important crop plant.