Keith Adams

Professor

Relevant Thesis-Based Degree Programs

 
 

Graduate Student Supervision

Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

Gene expression and alternative splicing responses to abiotic stress in sunflower and canola (2021)

Understanding the genetics and physiology of plant responses to abiotic stresses may offer insights into how crops might be modified to better avoid or tolerate such stresses. For my dissertation, I studied transcriptional (gene expression) and post-transcriptional (alternative splicing - AS) responses of canola and sunflower to several different abiotic stresses. First, I generated transcriptome data for canola, Brassica napus L., under heat, cold, and drought stress conditions. Brassica napus is an allotetraploid crop plant derived from B. rapa (AT) and B. oleracea (CT). Therefore, I focused on changes in gene expression and AS between homoeologous pairs of genes. I identified overall AT subgenome biases in gene expression and CT subgenome biases in the extent of alternative splicing under all three stress treatments. My results suggest that divergence in gene expression and AS patterns between duplicated genes may increase the flexibility of polyploid crops when responding to abiotic stressors.Second, I investigated drought responsive non-additive expression and AS events in parental and hybrid sunflower (Helianthus annuus L.) cultivars. Sunflower is a hybrid crop, and I employed well-characterized maternal and paternal lines and their F1 hybrid for this experiment. I showed that the expression of genes that were missing one or the other parental lines was complemented in the hybrid in both the control and drought treatment, offering a straightforward explanation for hybrid vigor or heterosis. Heterosis under drought stress was further associated with down-regulation of unnecessary metabolic pathways, via both a reduction in gene expression levels and an increase in non-functional splice forms.Lastly, using mixed model and gene co-expression network approaches, I found that up-regulation of ethylene-responsive transcription factors and down-regulation of metabolic, energy production, and photosynthesis-related pathways occurred in both the resistant and susceptible sunflower cultivated lines, and thus represent general responses to flooding stress. Greater flooding tolerance of the resistant line was associated with earlier and stronger up-regulation of the alcohol fermentation pathway and more rapid recovery of pre-flooding gene expression levels. Thus, adjustments to the timing of gene expression responses appear critical for establishing flooding stress tolerance in sunflowers.

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Fates of duplicated genes: sub-localization, intracellular gene transfer, and concerted evolution (2018)

Gene duplication is a major contributor to genome evolution. There are several evolutionary fates of duplicated genes, such as retention with redundancy, subfunctionalization and neofunctionalization. I proposed and characterized examples of three new models here. The first is duplication of an alternatively spliced gene with dual-targeted products, followed by partitioning of the splice forms between the duplicates so that the products of each duplicate are sub-localized. I report the plastid ascorbate peroxidase (cpAPX) genes as an example of sub-localization. I show angiosperms typically have one cpAPX gene that generates both thylakoidal tAPX and stromal sAPX through alternative splicing. I then identified several independent, lineage-specific sub-localization events with paralogs of specialized tAPX and sAPX. I determined that the sub-localization happened through two types of sequence evolution patterns. Second, I show an unreported type of duplicative intracellular transfer: transfer of a nuclear gene to the mitochondrial genome and transcription of the gene. The transcribed orf164 gene in the mitochondrial genome of several Brassicaceae species is derived from a nuclear gene that codes for an auxin responsive protein. Third, I studied POLYCOMB REPRESSIVE COMPLEX2 (PRC2) in Brassicaceae to demonstrate concerted divergence of simultaneously duplicated genes whose products function in the same complex. The VERNALIZATION (VRN)-PRC2 complex contains VRN2 and SWINGER (SWN), and both genes were duplicated during a whole-genome duplication to generate FERTILIZATION INDEPENDENT SEED2 (FIS2) and MEDEA (MEA), which function in the Brassicaceae-specific FIS-PRC2 complex that regulates reproductive development. I found that FIS2 and MEA have correlated reproductive-specific expression patterns that are derived from the broadly expressed VRN2 and SWN. In vegetative tissues of Arabidopsis thaliana, repressive methylation marks are enriched in FIS2 and MEA, whereas active marks are associated with their paralogs. I detected comparable accelerated amino acid substitution rates in FIS2 and MEA but not in their paralogs. These lines of evidence indicate that FIS2 and MEA have diverged in concert, resulting in functional divergence of the PRC2 complexes in Brassicaceae. Overall, the three projects provide new insights into the retention and divergence of duplicated genes.

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Evolution of duplicated non-coding RNAs in plants (2017)

Non-coding RNAs (ncRNAs) consist of microRNAs, lincRNAs (long intergenicnon-coding RNA), rRNAs, tRNAs and the RNAs from other types of genes that do nothave the potential to be protein-coding. Non-coding RNAs play various roles in cellularprocesses. Gene duplication is a major force in gene evolution and the evolution ofduplicated protein-coding genes has been studied extensively. Whether the sameevolutionary principles hold true for ncRNAs, especially lincRNAs, is still poorlyunderstood particularly in plants. I characterized the effects of the change in microRNAbinding sites on the divergence of multiple types of duplicated genes in Arabidopsisthaliana and Brassica rapa (Chapter 2). I found that the vast majority of duplicated genesshowed divergence in their microRNA binding sites that could be associated with theirexpression and functional divergence. To better understand the evolutionary dynamics oflincRNAs in plants, I analyzed the sequence evolution of lincRNAs from five species(Arabidopsis thaliana, Oryza sativa ssp. japonica, Zea mays, Medicago truncatula andSolanum lycopersicum) across 55 plant genomes (Chapter 3). My analyses revealed thatlincRNAs show more rapid sequence divergence compared with protein-coding genesand microRNAs. I also analyzed the expression conservation of lincRNAs betweenclosely related species and showed rapid expression evolution of lincRNAs. I alsoidentified a considerable number of conserved regions in the sequence of lincRNAs thatare under stronger selection constraints than surrounding regions. To investigate the roleof gene duplication in the evolution of plant lincRNAs, I identified duplicated lincRNAsiiiin several plant species (Chapter 4). I compared the expression patterns betweenduplicated lincRNAs using RNA-seq data from multiple tissue types and developmentalstages, revealing extensive expression divergence of lincRNAs. Finally, I studied theeffects of polyploidy and abiotic stress on the expression of lincRNAs in diploid andpolyploid Brassica species (Chapter 5). My results showed extensive divergence of theexpression of lincRNAs after polyploidy and in response to different stresses. This thesisprovides new insights into lincRNA evolution and fates of lincRNAs after duplication inflowering plants.

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The Evolution of Alternative Splicing After Polyploidy (2016)

Gene and genome duplications have made major contributions to the genomes of eukaryotes. Alternative splicing modulates gene expression and alters protein function. First, I examine alternative splicing patterns in the allopolyploid Brassica napus, revealing that the genome-wide trends of alternative splicing in duplicated genes of an evolutionarily new allotetraploid plant are very similar overall to those found in Arabidopsis thaliana. Within Brassica napus, I show that the alternative splicing patterns of the reunited homeologs are not well conserved, highlighting that alternative splicing is a rapidly evolving aspect of gene expression. Second, using Arabidopsis thaliana, I investigated the divergence of alternative splicing between paralogs, revealing about 30% qualitative conservation of alternative splicing events. I determined that qualitatively conserved events most often are not quantitatively conserved, indicating either incomplete divergence or specialization. I examined the duplicate gene pair of CCA1/LHY in detail, showing a case of subfunctionalization of alternative splicing after gene duplication that has implications for the cold response pathway of A. thaliana. By analyzing a transcriptome data set from nonsense mediated decay mutants, I showed that alternative splicing mediated nonsense mediated decay has significantly diverged between both pairs of whole genome and pairs of tandem duplicates. Third, I investigated the immediate effects of allopolyploidzation on gene expression and alternative splicing using three resynthesized Brassica napus lines. Many of the effects of allopolyploidization are repeatable, however some changes to gene expression and alternative splicing are unique to an instance of polyploidy. In all three polyploids surveyed, intron retention events that changed their frequency did so in an overwhelmingly negative fashion (i.e. the levels of alternatively spliced transcripts went down) and the majority of these changes were parallel between polyploids. Other classes of alternative splicing events showed a far more balanced set of changes in response to polyploidy. Natural B. napus showed significantly more increases in intron retention frequency vs. the parental species than any of the resynthesized lines. I assert that much of the changes in levels of alternatively spliced transcripts can be attributed the stochastic nature of polyploidization.

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Evolution of duplicate gene sequences, expression patterns, and functions in the Brassicaceae and other rosids (2011)

Duplicated genes are considered as raw materials for evolutionary innovations. They are common in eukaryotic genomes, particularly in plants due to the high incidence of whole genome duplication. Thus, understanding the factors that contribute to the retention of duplicatedgenes is a fundamental topic in evolutionary biology. I tackle this topic by examining howreciprocal expression (RE) among different organ and tissue types, as well as protein subcellularrelocalization (PSR), contributes to the retention of duplicated genes. From analyses ofmicroarray data across 83 different organ/cell types and developmental stages in Arabidopsisthaliana, I determined that more than 30% of duplicate pairs showed RE patterns (chapter 2).Reconstructing their ancestral expression pattern, more RE cases resulted from gain of a newexpression pattern (neofunctionalization) than from partitioning of ancestral expression patterns(subfunctionalization), with pollen being a common location for expression gain (chapter 2).During the analysis on RE, I found a dramatic example of neofunctionalization for a pair ofprotein kinase genes, SSP and BSK1, in the Brassicaceae (chapter 3). BSK1 and SSP have opposite expression patterns in pollen compared with all other parts of the plant. I determined that BSK1 retains the ancestral expression pattern and function and that the ancestral function of SSP was lost by deletions in the kinase domain. I revealed that SSP changed its function from a component of the brassinosteroid signaling pathway to being a paternal regulator ofembryogenesis. I also found that two reciprocally expressed duplicated gene pairs, a peroxidasegene pair and a CDPK gene pair, in Brassicaceae showed PSR and evidence for neofunctionalization (chapter 2). To better understand how PSR can contribute to the retention of duplicated genes, I focused on a particular example for a pair of the chloroplast-origin ribosomal protein S13 (rps13) genes in rosids (chapter 4). One encodes chloroplast-imported RPS13 (nucp rps13), while the other encodes mitochondria-imported RPS13 (numit rps13). I provided evidence that numit rps13 genes have experienced adaptive and convergent evolution. My thesisprovides important insights into the evolutionary importance of RE and PSR on the retention ofduplicated genes in plants.

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Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

Gene duplication and expression of specialized metabolic pathway genes in Cannabis sativa (2021)

Cannabis sativa is now widely cultivated for recreational and medicinal markets (drug-type cannabis) in addition to fiber and grain for materials and foodstuffs (fiber-type cannabis). Cannabis often is grown for its female flowers, which are highly concentrated with the specialized metabolites cannabinoids and terpenes. These compounds are specifically found in glandular trichomes, which are abundant on the surfaces of maturing cannabis flowers. However, both the abundance and identity of specialized metabolites can vary considerably across different cannabis varieties. Drug-type cannabis commonly contains the cannabinoid, THC, and fiber-type cannabis contains CBD. In this research, 24 gene families from the biochemical pathways responsible for cannabinoid and terpenoid production were analyzed across five different genomes of Cannabis sativa, containing a diversity of chemical and physical phenotypes. Orthologous genes from hops and three other Rosales species also were included. Gene duplication patterns and copy number variation were investigated using phylogenetic trees to define the evolutionary patterns within these biochemical pathways. Additionally, the duplicated genes within these pathways were analyzed for gene expression in several organ types of the Purple Kush variety. When comparing the terpenoid and cannabinoid pathways, both the non-mevalonate (MEP) and mevalonate (MVA) pathways contained fewer duplicated genes than the genes involved in cannabinoid biosynthesis. The evolutionary origins of the olivetolic acid cyclase (OAC) and aromatic prenyltransferase (APT) genes were revealed when comparing with hops and other closely related species. The gene expression analysis of the Purple Kush cultivar indicated that genes involved in both terpenoid and cannabinoid pathways were expressed highest in flowers. However, the number of expressed copies and expression levels varied among genes, and different copies are expressed in different organ types. Overall, this thesis provides insights to the evolutionary histories and gene expression patterns of the biochemical pathways involved in cannabinoid and terpenoid biosynthesis of Cannabis sativa.

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The effects of abiotic stress on isoform composition in polyploid Brassica napus (2021)

Polyploidy has played a major role throughout the evolution of plants and has long been considered a powerful driver of evolution across a broad range of plant lineages. Polyploidization events have occurred many times during the evolution of flowering plants (angiosperms). Following a polyploidization event, a set of duplicated genes is created which can diverge in function or new functions can evolve. Brassica napus, an allopolyploid derived from the hybridization of B. rapa and B. oleracea, more commonly known as canola, serves as an excellent model to study the complexities between duplicate gene pairs, also known as homeologous pairs. One of these complexities is the process of alternative splicing (AS) by which precursor mRNAs from multiexon genes are spliced to form mature mRNAs producing a vast repertoire of isoforms. The effects of abiotic stress conditions on isoform diversity and variability across homeologous pairs has received little attention.We conducted a global isoform sequencing analysis of Brassica napus using long-read sequencing of plants subjected to heat and cold stress. Analysis of AS events reveal a heat-responsive increase in the number of AS events. Furthermore, we discovered that cold stress reduces the number of isoforms produced by a given gene, whereas heat stress increases the number of isoforms produced by a given gene. This heat-responsive increase in the number of isoforms produced was paired with the observation that heat-stress also induces a higher number of transcripts predicted to be likely targets of nonsense-mediated decay (NMD), a common mechanism by which AS exerts transcriptional regulation. Our analysis also revealed that across homeologous pairs, C homeologs are more likely to produce more isoforms relative to A homeologs, across all three conditions tested. These shifted isoform distributions do not lead to shifted distributions in the predicted likelihood of NMD-targeting. In all, our analysis reveals opposing shifts in isoform composition in response to cold and heat stress as well as skewed isoform distributions across subgenomes, in which C homeologs are more likely to produce more isoforms relative to A homeologs, across each of the conditions we tested.

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Analysis of multiple Brassica transcriptomes reveals subgenome dominance in the response of Brassica napus to Sclerotinia sclerotiorum (2020)

Whole-genome duplication (WGD; polyploidy) events have played an extensive role in the evolution of flowering plants. The sudden doubling of genetic material can expedite rapid novel changes to polyploid transcriptomes. For example, polyploids formed via an interspecific hybridization of closely related species, known as allopolyploids, can exhibit inconsistent expression patterns between their parental genome. These incipient disparities in parental gene dosage can have profound effects on the transcriptome of newly formed polyploids, which in turn can influence their response to environmental stressors. In particular, the extent to which the transcriptomic shock of polyploidization modulates the biotic stress response of plant species remains a nascent topic in polyploidy research. To elicit such a response, I subjected both natural and newly formed lines of Brassica napus to pathogen infection with the fungal necrotroph Sclerotinia sclerotiorum. To understand the origin of subgenome divergence in the newly formed polyploid, I also performed infections on the diploid parents of the resynthesized B. napus, Brassica rapa and Brassica oleracea. RNA-seq analyses of these pathosystems revealed wide-spread divergence between polyploid subgenomes in terms of both constitutive gene expression and alternative splicing patterns. This manifested in a global expression bias towards the B. oleracea-derived (C) subgenome among both polyploid hosts, enhanced by widespread non-parental down-regulation of the B. rapa-derived (A) homeolog. In the resynthesized B. napus specifically, this resulted a disproportionate C subgenome contribution to plant innate immunity and pathogen defense response, characterized by biases in both transcript expression level and the proportion of induced genes.

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Divergence in alternative splicing patterns between duplicated gene pairs in polyploid Brassica napus (2010)

Polyploidy is the process of genome doubling that gives rise to organisms with multiple sets of chromosomes. Expression patterns and levels of genes duplicated by polyploidy, termed homeologs, can change and gene silencing can occur after polyploidy. Alternative splicing (AS) creates multiple mature mRNAs from a single type of precursor mRNA. AS can change the level of gene expression by degradation of transcripts with premature stop codons, as well as create new protein isoforms. Little is known about how AS changes after a polyploidization event, either within a few generations after polyploidy or over evolutionary time, and what effects AS changes have on gene expression in polyploids. In this project, the evolution of alternative splicing patterns after genome duplication in allotetraploid Brassica napus and a synthetic allotetraploid B. napus was examined by RT-PCR assays of a set of 31 duplicated genes. Since genes can show different patterns of AS in different organ types and under different abiotic stresses, two different organ types (leaf and cotyledon), and two different abiotic stresses (heat and cold) were used. Comparing the AS patterns between the two homeologs in B. napus revealed that 18% of the gene pairs show AS in only one homeolog. In contrast 33% of the gene pairs in the synthetic allotetraploid showed AS in only one homeolog. Gene silencing was observed for 6% and 9% of genes in B. napus and synthetic B. napus, respectively. These results indicate that there are many changes in AS in both the synthetic B. napus and natural B. napus after polyploidy, but more AS changes occurred in the synthetic polyploid. The PASTICCINO gene showed partitioning of two AS events between the homeologs in the synthetic allopolyploid, suggesting subfunctionalization of AS forms. Results from this project indicate that AS patterns can change rapidly after polyploidy and suggest that changes in AS patterns are a major phenomenon in allopolyploids.

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Evolution and neofunctionalization of imprinted genes after duplication in brassicaceae (2013)

Plant genomes have large numbers of duplicated genes. After duplication one duplicate can acquire a new function or expression pattern, referred to as neofunctionalization. Some duplicated genes are imprinted, where only one allele is expressed depending on its parental origin. I hypothesized that duplicated imprinted genes frequently show an accelerated rate of amino acid sequence evolution and have a new expression pattern compared with their paralogs, which together are suggestive of neofunctionalization. I first studied four imprinted genes in Arabidopsis, FIS2, MPC, FWA, and HDG3 that have flower and/or seed specific expression. I found that they all have considerably accelerated rates of sequence evolution compared to their paralogs. To determine the ancestral expression pattern I assayed expression patterns in outgroup species, the results of which strongly suggested that the imprinted genes have acquired a novel organ-specific expression pattern restricted to flowers and/or seeds. Using data from recent large-scale identification studies of imprinted genes, I detected by phylogenetic tree analyses 133 imprinted genes that arose from gene duplication events in Brassicaceae. Analyses of 48 alpha whole genome duplicated gene pairs indicated that many imprinted genes show an accelerated rate of amino acid changes compared to their paralogs. Analyses of microarray data indicated that many imprinted genes have expression patterns restricted to flowers and/or seeds, compared with their broadly-expressed paralogs. Both the accelerated sequence rate evolution and the new expression pattern in the imprinted genes suggest that after evolutionarily recent duplication events, imprinted genes frequently underwent neofunctionalization. In particular, neofunctionalization of the FIS2 gene has led to a change in the mechanism of regulating seed development in Brassicaceae. Multiple lines of evidence, when considered together, are highly suggestive of many origins of imprinting in Brassicaceae. This study reveals that the origin of genetic imprinting can arise over short evolutionary time periods and gene duplication serves as an important factor generating imprinted genes.

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Evolutionary Conservation of Long Intergenic Non-coding RNA Genes in Arabidopsis (2013)

Long intergenic non-coding RNA (lincRNA) genes are a poorly studied class of transcripts, particularly in plants. Because of the low levels of expression, high tissue speci- city, and rapid rate of evolution of lincRNA transcripts, the discovery and functionalannotation of these molecules is a signi cant challenge. Here, I report the annotationof 201 new lincRNA transcripts in Arabidopsis thaliana discovered using the results of asingle RNA-seq experiment of a normalized library. Using these sequences, along withthe 6 480 lincRNA genes annotated by Liu et al. (2012), I performed a pairwise sequence alignment experiment with the genomes of 22 plant species in order to discoverhighly conserved sequences within lincRNA loci. Of the 6 681 lincRNA sequences examined, 3 374 have highly conserved sequences supported by multiple genomic alignmentsto other species. Six of these show evidence of ongoing reduced sequence rate evolutionwhen single-nucleotide variant data from the recent evolutionary history of Arabidopsisthaliana. The rate of retention of these conserved regions within the Brassicaceae suggests a much higher rate of sequence turnover in lincRNA genes compared with proteincoding genes. Structural variant data from 80 di erent A. thaliana ecotypes suggeststhat lincRNA genes su er deletions of the entire locus from the genome with appreciablefrequency: 570 of the lincRNA loci examined are entirely missing from at least one A.thaliana strain. These results suggest an intriguing mixture of rapid sequence evolutionwith short, highly-conserved islands in lincRNA genes.

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Fates of genes after duplication: sublocalization and regulatory neofunctionalization (2013)

Gene duplication has supplied the raw material for novel gene functions and evolutionary innovations in plants. Duplicated genes can have different fates over time such as neofunctionalization and subfunctionalization. Sublocalization, which is a type of subfunctionalization based on protein subcellular relocalization, happens when the products of the duplicate genes are each directed to only one of two subcellular locations that were previously targeted by the single ancestral gene. The goals of the first part of my project were to study changes in protein subcellular localization (relocalization) after gene duplication by finding cases of sublocalization and further characterizing them from an evolutionary perspective. I found that sublocalization is a relatively uncommon phenomenon in plants as only two out of the seven gene families that I analyzed demonstrated cases of sublocalization. I identified and analyzed multiple cases of sublocalization of the APX and PP5 genes by doing RT-PCR experiments and then performing phylogenetic analyses and sequence rate analyses to further characterize the genes from an evolutionary perspective. Regulatory neofunctionalization involves changes in expression patterns of a gene after duplication. The goals for the second part of my thesis were to study expression patterns of duplicated genes in Arabidopsis thaliana and to analyze the selective forces acting on the genes of interest. I focused on eight pairs of duplicates that showed one copy broadly expressed and the other copy having expression only in certain organ types. By analyzing the expression patterns of the orthologs in outgroup species and selective forces acting on the sequences, I obtained evidence for potential neofunctionalization for a few cases. The results from my thesis provide new insights into the frequency and process of sublocalization of duplicated genes, as well as characterizing new examples of neofunctionalization of duplicated genes.

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Divergence in alternative splicing patterns between duplicated gene pairs in polyploid Brassica napus (2010)

Polyploidy is the process of genome doubling that gives rise to organisms with multiple sets of chromosomes. Expression patterns and levels of genes duplicated by polyploidy, termed homeologs, can change and gene silencing can occur after polyploidy. Alternative splicing (AS) creates multiple mature mRNAs from a single type of precursor mRNA. AS can change the level of gene expression by degradation of transcripts with premature stop codons, as well as create new protein isoforms. Little is known about how AS changes after a polyploidization event, either within a few generations after polyploidy or over evolutionary time, and what effects AS changes have on gene expression in polyploids. In this project, the evolution of alternative splicing patterns after genome duplication in allotetraploid Brassica napus and a synthetic allotetraploid B. napus was examined by RT-PCR assays of a set of 31 duplicated genes. Since genes can show different patterns of AS in different organ types and under different abiotic stresses, two different organ types (leaf and cotyledon), and two different abiotic stresses (heat and cold) were used. Comparing the AS patterns between the two homeologs in B. napus revealed that 18% of the gene pairs show AS in only one homeolog. In contrast 33% of the gene pairs in the synthetic allotetraploid showed AS in only one homeolog. Gene silencing was observed for 6% and 9% of genes in B. napus and synthetic B. napus, respectively. These results indicate that there are many changes in AS in both the synthetic B. napus and natural B. napus after polyploidy, but more AS changes occurred in the synthetic polyploid. The PASTICCINO gene showed partitioning of two AS events between the homeologs in the synthetic allopolyploid, suggesting subfunctionalization of AS forms. Results from this project indicate that AS patterns can change rapidly after polyploidy and suggest that changes in AS patterns are a major phenomenon in allopolyploids.

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Regulation of gene expression in invasive and non-invasive Compositae weeds (2010)

Gene expression divergence between populations has been linked to adaptive morphological evolution and is thought to be a factor in the invasive success of certain weedy plants. Understanding the genetic basis of these regulatory changes can identify genes that have been under selection during adaptation to a new environment or new species interactions. A high-throughput sequencing approach was used to study the regulatory basis (cis and/or trans) of gene expression differences between native and invasive populations of Cirsium arvense (Canada thistle) by exploring patterns of differential gene expression and sequence variation. Parent and hybrid allele-specific expression ratios were compared to infer the relative effects of cis- and trans-regulatory change. Genes differentially regulated in cis are considered candidate genes involved in adaptation or weediness because there is evidence for selection acting primarily on cis-regulatory variation. Illumina sequencing of cDNA libraries derived from parents and hybrid pools resulted in a total of 82,713,256 paired-end (2x100bp) reads and 83.4% of these were mapped to a reference C. arvense transcriptome of 88,374 unigene sequences. Expression analysis and variant (SNPs and Indel) calling was performed to score the nature of regulatory divergence for the first 900 contigs, representing ~1% of the total dataset. Of the 40 high-confidence cases, 7 showed cis-effects, 6 showed trans-effects, 9 had varying degrees of both cis and trans, and 18 showed non-intermediate hybrid effects. A set of contigs that had high similarity to 63 known or confirmed stress-related genes, previously identified in studies of sunflower and Canada thistle, was also assayed for allelic imbalance. Of these, 2 cases showed a cis-effect, 2 showed both cis- and trans-effects, and 2 revealed hybrid effects. Contig 23614, an auxin-response transcription factor, was differentially regulated due to cis-effects and has been previously confirmed as drought-stress gene in both sunflower and C. arvense. This research identifies changes in gene expression that are driven by differential selective pressures in native and invasive populations. It also advances our understanding of the nature of genetic changes that drive gene expression evolution.

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The genetic control of petal morphology in the sweet pea (Lathyrus odoratus) (2010)

The hooded mutant of sweet pea (Lathyrus odoratus) was characterized in order to better understand the genetic control of petal morphology in papilionoid legumes. The TCP identity gene CYCLOIDEA2 (CYC2) was shown to have different amplification patterns in the standard petals of hooded and wild-type sweet peas in RT-PCR experiments. Lathyrus CYC1 was also examined, but no differences were found between expression patterns in wild-type and hooded plants. Genome walking and additional sequencing revealed that the hooded phenotype was associated with a mutant allele of CYC2. To determine if the mutant allele of CYC2 is responsible for the hooded phenotype, a F2 segregation experiment was conducted. In a population of 118 plants, the hooded phenotype segregated with the mutant allele of CYC2 100% of the time, suggesting that the mutation in CYC2 is likely responsible for the hooded floral character. Scanning Electron Microscopy was conducted to determine if epidermal cell types could be used as micromorphological markers for petal identity. Cells on the adaxial surface of sweet pea petals can be used to distinguish petal identity. When mutant and wild-type flowers were compared, clear differences in cell type were observed. The standard petal in hooded flowers has taken on wing petal characteristics, indicating that the mutation is caused by a shift in petal identity. To examine localized growth patterns in wild-type and hooded flowers, microscopic grids were printed on the surfaces of sweet pea buds using a specialized inkjet apparatus. The deformation of the printed grid during growth allowed localized patterns of growth to be visualized. Wild-type standard petals have a more uniform rate of growth, whereas hooded standard petals show increased growth at their margins which may account for the overall differences in curvature between wild-type and hooded petals. The results of these analyses suggest that the hooded mutant of sweet pea is caused by a mutation in the CYC2 gene. CYC2 genes have been shown to play a role in establishing standard petal identity in the sweet pea and in a number of other legumes (Feng et al., 2006; Wang et al., 2008a).

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