Research interests in the Wendel lab encompass molecular and genome evolution, phylogenetics, and phenotypic evolution of higher plants. In our laboratory we use a diverse set of technologies and approaches to explore the manner in which genomes change over evolutionary time, as well as the relationship between these events and morphological change. We have a particular interest in the mysterious and common phenomenon of polyploidy, with a special focus on the cotton genus. The “wordle” below encapsulates our primary research foci, with brief descriptions of our primary projects following.
Cotton fiber and the domestication process
One of the exciting opportunities stimulated by the convergence of modern genomic approaches with other areas of biology is that of resolving the enigmatic processes by which new phenotypes arise. Using a well-developed model system from the cotton genus (Gossypium) and multiple genomic resources, we are using comparative approaches combined with advanced population development to will reveal the steps and complexities involved in transforming primitive trichomes to the economically important fibers of modern cotton cultivars.
Our goal is to understand the genetic causes and system-wide effects that underlie phenotypic change. An exciting dimension to our work is that it involves domestication at both the diploid and allopolyploid levels, permitting us to explore the possibility that polyploid formation created novel opportunities for phenotypic evolution. Also, because two different allopolyploid species were independently domesticated, we have an outstanding opportunity to evaluate parallelisms and convergences in the underlying genetic architecture associated with plant domestication and morphological evolution.
We are using a number of tools in this effort, including comparative transcriptomic and proteomic profiling. One example is that of Yoo and Wendel (2014; see publication page), where the most important cotton species, Gossypium hirsutum, was studied. This is an allopolyploid species (containing two different genomes) which initially was domesticated approximately 5000 years ago, and which primarily is grown for its single-celled seed fibers. Strong directional selection over the millennia was accompanied by transformation of the short, coarse, and brown fibers of wild plants into the long, strong, and fine white fibers of the modern cotton crop plant.
To explore the evolutionary genetics of cotton domestication, we conducted transcriptome profiling of developing cotton fibers from multiple accessions of wild and domesticated cottons. Comparative analysis revealed that the domestication process dramatically rewired the transcriptome, affecting more than 5,000 genes, and with a more evenly balanced usage of the duplicated copies arising from genome doubling. We identify many different biological processes that were involved in this transformation, including those leading to a prolongation of fiber elongation and a reallocation of resources toward increased fiber growth in modern forms. The data provide a rich resource for future functional analyses targeting crop improvement and evolutionary objectives.
Evolution of duplicate gene expression
One of the important realizations to emerge from numerous studies of polyploid plants is that polyploid creates massive alterations in gene expression. In every allopolyploid examined to date, some fraction of the duplicate gene pairs are 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. This can vary quantitatively and qualitatively among species, tissues, and even cell lines and single cells, and is a fundamental feature of allopolyploids.
In addition to this genome bias, polyploids almost always show expression level dominance, where, for a given gene, the total expression of homoeologs is statistically the same as only one of the polyploid parents. Originally described by Rapp et al. (2009) using cotton, this work has been confirmed and extended by Flagel & Wendel (2010), Yoo et al., (2013) and many others (see Grover et al., 2012 for perspective and review; all citations on publication page). Now that the phenomenon has been described, we are undertaking efforts to understand the mechanistic bases of both homoeolog bias and expression level dominance, using a combination of molecular biological tools including high throughput sequencing, monococcal nuclease assays, and chromatin immunoprecipitation. This work holds promise for helping us understand, for example, why some duplicate gene pairs are co-regulated, whereas other homoeolog pairs behave independently of one another.
Evolution of Duplicated Pathways and Networks
Polyploid speciation is exceptionally common in plants, often operating sympatrically to saltationally generate new lineages. The reunion between two diverged diploid genomes during hybridization and polyploidization sets in motion a wide spectrum of genetic and genomic phenomena whose mechanistic underpinnings remain mostly mysterious and that have largely unknown evolutionary consequences. To advance our understanding of the evolutionary response dynamics of genome doubling, we have focused on illustrating the consequences of allopolyploid speciation on pathways and networks, using the anthocyanin biosynthetic pathway and flowering time regulation systems. This research entails complementary experimental approaches, including genetic and phylogenetic characterization of pathway/network components, transcriptomic profiling of gene expression dynamics accompanying genome evolution, and quantitative comparison of metabolite accumulation in response to pathway duplication.
Because mRNA abundance and protein amounts often are poorly related, it is important to extend the evolutionary analysis of polyploidy and domestication to the proteomic level. Using comparative proteomic approaches, including two-dimensional gel electrophoresis (2-DE) and iTRAQ, we have characterized global protein changes corresponding to polyploidization and human-mediated selection under domestication. We have identified proteins differentially expressed during cotton fiber development in wild and domesticated accessions of both G. barbadense and G. hirsutum, and have discovered how these have been altered by domestication (see Hu et al. citations on publication page). These proteins become candidates for metabolic processes underlying cotton fiber development and domestication, and for future functional analyses that may yield insight into domestication and cotton improvement. A combined proteomic and transcriptomic analysis on cotton seed development in diploid and polyploid species is in progress.
New Cotton Species
Gossypium ekmanianum was resurrected as a 6th polyploid species by Krapovickas and Seijo in 2008, distinct from both G. hirsutum (AD1) and G. barbadense (AD2). Krapovickas and Seijo used morphology as a basis for this proposal, but no morphological characters were cited that clearly or cleanly distinguish G. ekmanianum from the other polyploid species. Using both cpDNA sequence/indel polymorphisms and nuclear gene sequences, we have confirmed that G. ekmanianum warrants recognition as a distinct species (Grover et al., 2014 on publication page).
In addition, some polyploid cotton accessions from the Wake Atoll (Wilkes and Peale Islands) previously considered to belong to AD1, have been determined to be highly divergent from the rest of that latter species. Present analyses of hundreds of nuclear gene sequences and of cpDNA indicate that this too should be recognized as a new (the 7th) polyploid cotton species, which we provisionally are naming Gossypium stephensii, in honor of the great cotton cytogeneticist, plant explorer, and evolutionary biologist S. G. Stephens (see Wendel and Goodman, 2011, PNAS memoirs).
Finally, we have just finished publishing the name G. anapoides, for a new species of K-genome cotton from the Kimberley region of NW Australia, which Wendel first discovered from a helicopter (!) during an aerial survey of the region in 1993.
Genome Size Evolution
Genome size (GS) in land plants varies by over 2000-fold and much of this diversity is the result of varying quantities of repetitive DNA, with very little arising from variation in genic content. The cotton genus provides an excellent opportunity to reveal the dynamics of genome size evolution (see Grover and Wendel, 2010, publication page) because it contains diploid species that vary three-fold in DNA content. To gain insights into the patterns and processes governing fluctuations in GS, we are using genomic re-sequencing data and representatives from each cotton genome group (A-G, K, and polyploid AD) to assess repetitive DNA evolution in a phylogenetic context. Ancestral states with regard to both GS and repetitive DNA content (down to individual repeat families) are being reconstructed to provide a robust evolutionary framework to study genome size evolution in Gossypium genus. In addition to investigating interspecific GS variation, we also are using re-sequencing data of multiple accessions of 4 different species (i.e., A2, D5, AD1, and AD2) to uncover patterns of repetitive element variation and evolution within species.
Centromere Evolution in Diploid and Polyploid Plants
In diploids centromere sequences typically become rapidly homogenized within and among chromosomes, but the evolutionary dynamics of this remain largely unexplored in allopolyploids. We intend to gain insight into the evolutionary genomics of centromeres (see Masonbrink et al., on publication page) and repetitive DNA, during diploid divergence and following polyploidy in the cotton genus. Our experimental protocol includes the use of an antibody to the CenH3 protein to precipitate centromeric DNA for sequencing in cotton allopolyploids and their proposed progenitor diploid species. Bioinformatic analyses, including the assembly of each centromere will provide insight into the rate, pattern, and mechanism of sequence dispersal and exchange between homologous and homoeologous chromosomes.
One underexplored dimension of allopolyploid evolution is cytonuclear interactions. Potential stoichiometric disruption caused by merging two nuclear genomes but only inheriting one set of progenitor organellar genomes (usually the maternal) suggests that cytonuclear accommodation is a necessary aspect of allopolyploid evolution. Our exploration of cytonuclear accommodation in allotetraploids of Gossypium has contributed initial understanding about how nuclear homoeologous genes (from both parental diploid species) encoding component subunits of one protein complex have evolved in the context of having their counterpart subunits encoded by genes from only one (the maternal) parent. The model protein complex we utilized is Rubisco (Ribulose 1,5-bisphosphate carboxylase/oxygenase), an essential enzyme in carbon fixation during photosynthesis, encoded by a nuclear rbcS multigene family and a single plastid rbcL gene. In Gossypium, we demonstrated their cytonuclear coordination at both genomic level and the transcriptional level. Further analysis in other angiosperm polyploid systems are being carried out to ask whether the coordination mechanisms are commonly employed by different polyploid lineages in plant kingdom (see Gong et al. citations on publication page).
Small RNA Evolution
Small RNAs are a diverse category of nuclear-encoded non-coding RNAs that play multiple, central functions in eukaryotic development, stress responses and many other biological processes. The major two types of small RNAs are microRNAs (miRNAs) and silencing RNAs (siRNAs). The major focus of this project is the role of and divergence among sRNAs during cotton diploid divergence, polyploid formation, and cotton domestication. Accordingly, millions of small RNA sequences from next generation sequencing have been generated and analyzed from respective A- and D- diploid cotton and AD1 (upland) wild and domesticated polyploid cotton species. Based on recently published A2 (G.arboreum) and D5 (G. raimondii) genome assemblies, the composition, expression and evolutionary origins of the miRNA families is being characterized. The expression of siRNAs and distribution pattern in both diploid, wild and domesticated cotton species are also being analyzed. Current analyses are mainly focused on miRNA characterization and expression determination in diploid and polyploid cotton species.