Jonathan F. Wendel

Genome evolution in polyploids

Polyploidy is a prominent process in plants and has been significant in the evolutionary history of vertebrates and other eukaryotes. In plants, interdisciplinary approaches combining phylogenetic and molecular genetic perspectives have enhanced our awareness of the myriad genetic interactions made possible by polyploidy. Here, processes and mechanisms of gene and genome evolution in polyploids are reviewed. Genes duplicated by polyploidy may retain their original or similar function, undergo diversification in protein function or regulation, or one copy may become silenced through mutational or epigenetic means. Duplicated genes also may interact through inter-locus recombination, gene conversion, or concerted evolution. Recent experiments have illuminated important processes in polyploids that operate above the organizational level of duplicated genes. These include inter-genomic chromosomal exchanges, saltational, non-Mendelian genomic evolution in nascent polyploids, inter-genomic invasion, and cytonuclear stabilization. Notwithstanding many recent insights, much remains to be learned about many aspects of polyploid evolution, including: the role of transposable elements in structural and regulatory gene evolution; processes and significance of epigenetic silencing; underlying controls of chromosome pairing; mechanisms and functional significance of rapid genome changes; cytonuclear accommodation; and coordination of regulatory factors contributed by two, sometimes divergent progenitor genomes. Continued application of molecular genetic approaches to questions of polyploid genome evolution holds promise for producing lasting insight into processes by which novel genotypes are generated and ultimately into how polyploidy facilitates evolution and adaptation.

Ribosomal ITS sequences and plant phylogenetic inference.

One of the most popular sequences for phylogenetic inference at the generic and infrageneric levels in plants is the internal transcribed spacer (ITS) region of the 18S-5.8S-26S nuclear ribosomal cistron. The prominence of this source of nuclear DNA sequence data is underscored by a survey of phylogenetic publications involving comparisons at the genus level or below, which reveals that of 244 papers published over the last five years, 66% included ITS sequence data. Perhaps even more striking is the fact that 34% of all published phylogenetic hypothesis have been based exclusively on ITS sequences. Notwithstanding the many important contributions of ITS sequence data to phylogenetic understanding and knowledge of genome relationships, a number of molecular genetic processes impact ITS sequences in ways that may mislead phylogenetic inference. These molecular genetic processes are reviewed here, drawing attention to both underlying mechanism and phylogenetic implications. Among the most prevalent complications for phylogenetic inference is the existence in many plant genomes of extensive sequence variation, arising from ancient or recent array duplication events, genomic harboring of pseudogenes in various states of decay, and/or incomplete intra- or inter-array homogenization. These phenomena separately and collectively create a network of paralogous sequence relationships potentially confounding accurate phylogenetic reconstruction. Homoplasy is shown to be higher in ITS than in other DNA sequence data sets, most likely because of orthology/paralogy conflation, compensatory base changes, problems in alignment due to indel accumulation, sequencing errors, or some combination of these phenomena. Despite the near-universal usage of ITS sequence data in plant phylogenetic studies, its complex and unpredictable evolutionary behavior reduce its utility for phylogenetic analysis. It is suggested that more robust insights are likely to emerge from the use of single-copy or low-copy nuclear genes.

Visualization and Interpretation of Plant Isozymes

Gel electrophoresis of proteins has become a standard and powerful research tool for application in a multitude of biological disciplines. One form of protein electrophoresis, isozyme analysis, has become particularly prominent in systematic and evolutionary biology as well as agronomy (Tanksley and Orton, 1983). Isozymes, or multiple molecular forms of enzymes, are enzymes that share a common substrate but differ in electrophoretic mobility (Markert and Moller, 1959). They are revealed when tissue extracts are subjected to electrophoresis in various types of gels and subsequently submersed in solutions containing enzyme-specific stains. Genetic analysis may indicate that some of the variant electromorphs are encoded by alternate alleles at a single locus, in which case the allelic products are termed allozymes (Prakash et al., 1969). Data retrieved from electrophoretic gels consist of the number and relative mobilities of various enzyme products, which with appropriate genetic analyses become transformed into single or multilocus genotypes for each individual analyzed. Reasons are many for the popularity of electrophoretic data (Avise, 1975; Gottlieb, 1977; Crawford, 1983), but foremost among these is that isozymes provide a series of readily scored, single-gene markers.

Genes duplicated by polyploidy show unequal contributions to the transcriptome and organ-specific reciprocal silencing.

Most eukaryotes have genomes that exhibit high levels of gene redundancy, much of which seems to have arisen from one or more cycles of genome doubling. Polyploidy has been particularly prominent during flowering plant evolution, yielding duplicated genes (homoeologs) whose expression may be retained or lost either as an immediate consequence of polyploidization or on an evolutionary timescale. Expression of 40 homoeologous gene pairs was assayed by cDNA-single-stranded conformation polymorphism in natural (1- to 2-million-yr-old) and synthetic tetraploid cotton (Gossypium) to determine whether homoeologous gene pairs are expressed at equal levels after polyploid formation. Silencing or unequal expression of one homoeolog was documented for 10 of 40 genes examined in ovules of Gossypium hirsutum. Assays of homoeolog expression in 10 organs revealed variable expression levels and silencing, depending on the gene and organ examined. Remarkably, silencing and biased expression of some gene pairs are reciprocal and developmentally regulated, with one homoeolog showing silencing in some organs and the other being silenced in other organs, suggesting rapid subfunctionalization. Duplicate gene expression was examined in additional natural polyploids to characterize the pace at which expression alteration evolves. Analysis of a synthetic tetraploid revealed homoeolog expression and silencing patterns that sometimes mirrored those of the natural tetraploid. Both long-term and immediate responses to polyploidization were implicated. Data suggest that some silencing events are epigenetically induced during the allopolyploidization process.

A rapid method for extraction of cotton (Gossypium spp. ) genomic DNA suitable for RFLP or PCR analysis.

Extraction of high-quality genomic DNA fromGossypium (cotton) species is difficult due to high levels of polysaccharide, oxidizable quinones, and other interfering substances. We describe a procedure that consistently permits isolation of cotton genomic DNA of satisfactory size and quality for RFLP and PCR analysis, as well as for most routine cloning applications. Several antioxidants, phenol-binding reagents, and phenol oxidase inhibitors are used throughout the procedure, and most polysaccharides are eliminated early in the procedure by isolation of nuclei.

Genetics of Plant Isozymes

The ability to observe allelic variation at isozyme loci has revolutionized research in the fields of biochemical genetics, population genetics, and evolution. This variation, called allozymic polymorphism, has been used in plants to examine genetic processes at every stage of the life cycle and to ascertain genetic diversity in all major crops as well as many other species. Yet the potential for using allozymes as genetic markers was not immediately predicted when isozyme variability was initially described (Hunter and Marker, 1957; Markert and Moller, 1959). In fact, the extent and prevalence of allozyme polymorphism was a rather disconcerting surprise to evolutionary biologists. Classical evolutionary theory had predicted that the most “efficient” form of an enzyme should, over time, become predominant in isolated populations, with an occasional rare allele produced through mutation. The discovery in many populations of relatively high levels of polymorphism at isozyme loci has forced a major reconsideration of evolutionary theory (Kimura and Crow, 1964; Koehn et al., 1983; Kimura, 1983).

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.

Polyploidy and the Evolutionary History of Cotton

Abstract The cotton genus ( Gossypium ) includes approximately 50 species distributed in arid to semi-arid regions of the tropic and subtropics. Included are four species that have independently been domesticated for their fiber, two each in Africa–Asia and the Americas. Gossypium species exhibit extraordinary morphological variation, ranging from herbaceous perennials to small trees with a diverse array of reproductive and vegetative characteristics. A parallel level of cytogenetic and genomic diversity has arisen during the global radiation of the genus, leading to the evolution of eight groups of diploid ( n =13) species (genome groups A–G, and K). The evolutionary history of the genus included multiple episodes of trans-oceanic dispersal, invasion of new ecological niches, and a surprisingly high frequency of natural interspecific hybridization among lineages that are presently both geographically isolated and intersterile. Recent investigations have clarified many aspects of this history, including relationships within and among the eight genome groups, the domestication history of each of the four cultivated species, and the origin of the allopolyploid cottons. Data implicate an origin for Gossypium 5–15 million years ago (mya) and a rapid early diversification of the major genome groups. Allopolyploid cottons appear to have arisen within the last million years, as a consequence of trans-oceanic dispersal of an A-genome taxon to the New World followed by hybridization with an indigenous D-genome diploid. Subsequent to formation, allopolyploids radiated into three modern lineages, including those containing the commercially important species G. hirsutum and G. barbadense . Genome doubling has led to an array of molecular genetic interactions, including inter-locus concerted evolution, differential rates of genomic evolution, inter-genomic genetic transfer, and probable alterations in gene expression. The myriad underlying mechanisms are also suggested to have contributed to both ecological success and agronomic potential.

Phylogenetic Incongruence: Window into Genome History and Molecular Evolution

The field of systematic biology has been revitalized and transformed during the last few decades by the confluence of phylogenetic thinking with ready access to the tools of molecular biology. Indeed, the title of this volume and the fact that it is already in its second edition offers ample testimony to the impact that molecular approaches have had on efforts to reconstruct the phylogenetic history of plants. Concomitant with the proliferation of molecular tools has been a growing awareness that reliance on a single data set may often result in insufficient phylogenetic resolution or misleading inferences. Accordingly, it is an increasingly widespread practice to apply multiple data sets to a common group of taxa. One of the consequences of analyzing multiple data sets is that the phylogenies inferred may differ from each other in one or more details. This phylogenetic incongruence is not rare; to the contrary, it is almost the rule rather than the exception, being evident to varying degrees.

Evolutionary Genetics of Genome Merger and Doubling in Plants

Polyploidy is a common mode of evolution in flowering plants. The profound effects of polyploidy on gene expression appear to be caused more by hybridity than by genome doubling. Epigenetic mechanisms underlying genome-wide changes in expression are as yet poorly understood; only methylation has received much study, and its importance varies among polyploids. Genetic diploidization begins with the earliest responses to genome merger and doubling; less is known about chromosomal diploidization. Polyploidy duplicates every gene in the genome, providing the raw material for divergence or partitioning of function in homoeologous copies. Preferential retention or loss of genes occurs in a wide range of taxa, suggesting that there is an underlying set of principles governing the fates of duplicated genes. Further studies are required for general patterns to be elucidated, involving different plant families, kinds of polyploidy, and polyploids of different ages.