Corrinne E. Grover

Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres

Polyploidy often confers emergent properties, such as the higher fibre productivity and quality of tetraploid cottons than diploid cottons bred for the same environments. Here we show that an abrupt five- to sixfold ploidy increase approximately 60 million years (Myr) ago, and allopolyploidy reuniting divergent Gossypium genomes approximately 1–2 Myr ago, conferred about 30–36-fold duplication of ancestral angiosperm (flowering plant) genes in elite cottons (Gossypium hirsutum and Gossypium barbadense), genetic complexity equalled only by Brassica among sequenced angiosperms. Nascent fibre evolution, before allopolyploidy, is elucidated by comparison of spinnable-fibred Gossypium herbaceum A and non-spinnable Gossypium longicalyx F genomes to one another and the outgroup D genome of non-spinnable Gossypium raimondii. The sequence of a G. hirsutum AtDt (in which ‘t’ indicates tetraploid) cultivar reveals many non-reciprocal DNA exchanges between subgenomes that may have contributed to phenotypic innovation and/or other emergent properties such as ecological adaptation by polyploids. Most DNA-level novelty in G. hirsutum recombines alleles from the D-genome progenitor native to its New World habitat and the Old World A-genome progenitor in which spinnable fibre evolved. Coordinated expression changes in proximal groups of functionally distinct genes, including a nuclear mitochondrial DNA block, may account for clusters of cotton-fibre quantitative trait loci affecting diverse traits. Opportunities abound for dissecting emergent properties of other polyploids, particularly angiosperms, by comparison to diploid progenitors and outgroups.

PCR-mediated recombination in amplification products derived from polyploid cotton

Abstract  PCR recombination describes a process of in vitro chimera formation from non-identical templates. The key requirement of this process is the inclusion of two partially homologous templates in one reaction, a condition met when amplifying any locus from polyploid organisms and members of multigene families from diploid organisms. Because polyploids possess two or more divergent genomes (”homoeologues”) in a common nucleus, intergenic chimeras can form during the PCR amplification of any gene. Here we report a high frequency of PCR-induced recombination for four low-copy genes from allotetraploid cotton (Gossypium hirsutum). Amplification products from these genes (Myb3, Myb5, G1262 and CesA1) range in length from 860 to 4,050 bp. Intergenomic recombinants were formed frequently, accounting for 23 of the 74 (31.1%) amplicons evaluated, with the frequency of recombination in individual reactions ranging from 0% to approximately 89%. Inspection of the putative recombination zones failed to reveal sequence-specific attributes that promote recombination. The high levels of observed in vitro recombination indicate that the tacit assumption of exclusive amplification of target templates may often be violated, particularly from polyploid genomes. This conclusion has profound implications for population and evolutionary genetic studies, where unrecognized artifactually recombinant molecules may bias results or alter interpretations.

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.

Targeted sequence capture as a powerful tool for evolutionary analysis1

Next-generation sequencing technologies (NGS) have revolutionized biological research by significantly increasing data generation while simultaneously decreasing the time to data output. For many ecologists and evolutionary biologists, the research opportunities afforded by NGS are substantial; even for taxa lacking genomic resources, large-scale genome-level questions can now be addressed, opening up many new avenues of research. While rapid and massive sequencing afforded by NGS increases the scope and scale of many research objectives, whole genome sequencing is often unwarranted and unnecessarily complex for specific research questions. Recently developed targeted sequence enrichment, coupled with NGS, represents a beneficial strategy for enhancing data generation to answer questions in ecology and evolutionary biology. This marriage of technologies offers researchers a simple method to isolate and analyze a few to hundreds, or even thousands, of genes or genomic regions from few to many samples in a relatively efficient and effective manner. These strategies can be applied to questions at both the infra- and interspecific levels, including those involving parentage, gene flow, divergence, phylogenetics, reticulate evolution, and many more. Here we provide a brief overview of targeted sequence enrichment, and emphasize the power of this technology to increase our ability to address a wide range of questions of interest to ecologists and evolutionary biologists, particularly for those working with taxa for which few genomic resources are available.

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.

A Phylogenetic Analysis of Indel Dynamics in the Cotton Genus

Genome size evolution is a dynamic process involving counterbalancing mechanisms whose actions vary across lineages and over time. Whereas the primary mechanism of expansion, transposable element (TE) amplification, has been widely documented, the evolutionary dynamics of genome contraction have been less thoroughly explored. To evaluate the relative impact and evolutionary stability of the mechanisms that affect genome size, we conducted a phylogenetic analysis of indel rates for 2 genomic regions in 4 Gossypium genomes: the 2 coresident genomes (A(T) and D(T)) of tetraploid cotton and its model diploid progenitors, Gossypium arboreum (A) and Gossypium raimondii (D). We determined the rates of sequence gain or loss along each branch, partitioned by mechanism, and how these changed during species divergence. In general, there has been a propensity toward growth of the diploid genomes and contraction in the polyploid. Most of the size difference between the diploid species occurred prior to polyploid divergence and was largely attributable to TE amplification in the A/A(T) genome. After separating from the true parents of the polyploid genomes, both diploid genomes experienced slower sequence gain than in the ancestor, due to fewer TE insertions in the A genome and a combination of increased deletions and decreased TE insertions in the D genome. Both genomes of the polyploid displayed increased rates of deletion and decreased rates of insertion, leading to a rate of near stasis in D(T) and overall contraction in A(T) resulting in polyploid genome contraction. As expected, TE insertions contributed significantly to the genome size differences; however, intrastrand homologous recombination, although rare, had the most significant impact on the rate of deletion. Small indel data for the diploids suggest the possibility of a bias as the smaller genomes add less or delete more sequence through small indels than do the larger genomes, whereas data for the polyploid suggest increased sequence turnover in general (both as small deletions and small insertions). Illegitimate recombination, although not demonstrated to be a dominant mechanism of genome size change, was biased in the polyploid toward deletions, which may provide a partial explanation of polyploid genomic downsizing.

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.

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.

Assessing the monophyly of polyploid Gossypium species

The origin and monophyly of the polyploid cotton (Gossypium) species has been largely accepted, despite the lack of explicit phylogenetic evidence. Recent studies in other polyploid systems have demonstrated that multiple origins for polyploid species are much more common than once thought, raising the possibility that Gossypium polyploids also had multiple origins, as postulated by some authors. To test the monophyly of polyploid cotton, we sequenced a 2.8-kb intergenic region from all diploid species belonging to the genome groups from which the polyploid originates. The resulting phylogenetic analyses strongly support a single origin of polyploid cotton involving a D-genome ancestor related to Gossypium raimondii and an A-genome ancestor that was sister to both extant A-genome species.

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.