Polyploidy, or whole genome duplication, is rampant among both extant and ancient flowering plant species. Whole genome duplications create simultaneous copies of all genes contained within a genome as well as associated regulatory sequences. These duplication and the subsequent deletions of redundant coding and noncoding sequence both shape the natural evolution of plant genomes and provide a unique opportunity for researchers to characterize the regulatory sequences which determine when, in which cells and in what quantities the mRNA encoded for by particular genes will be produced.

This dissertation describes a model for explaining both bias in gene loss between parental subgenomes and the escape from preferential retention of duplicated genes between sequential whole genome duplications. Bias in gene deletion between individual duplicated segments had been previously observed by the publication of the sorghum and maize genomes provided an opportunity to demonstrate this bias was a consistent mark distinguishing whole pairs of ancestral chromosomes, and that ongoing gene loss remains consistently biased between high and low gene loss subgenomes millions of generations after a whole genome duplication. Bias in both ancestral and ongoing gene loss is shown to be correlated with biased gene expression between parental subgenomes with genes on the low gene loss subgenome tending to show higher expression levels than duplicate copies of the same genes on the high gene loss subgenome. This phenomena, originally referred to as genome dominance, although the literature has since become somewhat confused, provides an explanation both for biased gene loss between parental subgenomes and for the escape of deletion-resistant genes from the ratchet of ever increasing copy numbers through continued whole genome duplications.

This dissertation also demonstrates the use of polyploid lineage - in this case maize - as a deletion machine to rapidly characterize the function of regulatory sequences shared by orthologous genes within a clade. It was possible to develop testable hypothesis about the specific function of individual regulatory sequences by combining conserved noncoding sequence sequence datasets, noncoding sequence deletions identified using comparative genomics with analysis and visualization of gene expression data from diverse organs, tissues, and cell types. As a test of the accuracy of this method, a putative pollen specific enhancer of expression identified using expression data from maize was cloned from the orthologous sorghum gene and used to drive the expression of a reporter construct in Brachypodium distachyon. Polyploid deletion machines have the potential to radically accelerate the characterization of noncoding regulatory sequences, an area of genetics previously largely untouched by advances next generation sequencing technologies.

Abstract

Patterns of computed conserved noncoding sequence loss

following the paleopolyploidies in the maize and Brassica lineages

and their functional consequences

by

Sabarinath Subramaniam

Doctor of Philosophy in Plant Biology

University of California, Berkeley

Professor Michael Freeling, Chair

Following polyploidy, duplicate genes are often deleted, and if

they are not, then duplicate regulatory regions are sometimes lost.

By what mechanism is this loss, and what is the chance that such a

loss removes function? To explore these questions, we followed

individual Arabidopsis thaliana-Arabidopsis thaliana conserved

noncoding sequences (CNSs) into the Brassica ancestor, through a

paleohexaploidy and into Brassica rapa. Thus, a single

Brassicaceae CNS has six potential orthologous positions in

Brassica rapa; a single arabidopsis CNS has three potential

homeologous positions. We reasoned that a CNS, if present on a

singlet Brassica gene, would be unlikely to lose function compared

to a more redundant CNS, and this is the case. Redundant CNSs

go nondetectable often. Using this logic, each mechanism of CNS

loss was assigned a metric of functionality. By definition, proved

deletions do not function as sequence. Our results indicated that

CNSs that go nondetectable by base substitution or large insertion

are almost certainly still functional (redundancy does not matter

much to their detectability frequency), while, those lost by inferred

deletion or indels are about 75% likely to be nonfunctional.

Overall, an average nondetectable, once-redundant CNS > 30 bps

in length has a 72% chance of being nonfunctional, and that makes

sense because 97% of them sort to a molecular mechanism with

"deletion" in its description, but base substitutions do cause loss.

Similarly, proved-functional G-boxes go undetectable by deletion

82% of the time. Fractionation mutagenesis is a procedure that

uses polyploidy as a mutagenic agent to genetically alter RNA

expression profiles, and then to construct testable hypotheses as to

the function of the lost regulatory site. We show fractionation

mutagenesis to be a "deletion machine" in the Brassica lineage.

Transposons make up a sizable portion of most genomes, and most organisms have evolved mechanisms to silence them. In maize, silencing of the Mutator family of transposons is associated with methylation of the terminal inverted repeats (TIRs) surrounding the autonomous element and loss of mudrA expression (the transposase) as well as mudrB (a gene involved in insertional activity). We have previously reported that a mutation that suppresses paramutation in maize, mop1, also hypomethylates Mat elements and restores somatic activity to silenced MuDR elements. Here, we describe the progressive reactivation of silenced mudrA after several generations in a mop1 background. In mop1 mutants, the TIRA becomes hypomethylated immediately, but mudrA expression and significant somatic reactivation is not observed until silenced MuDR has been exposed to mop] for several generations. In subsequent generations, individuals that are heterozygous or wild type for the Mop1 allele continue to exhibit hypomethylation at Mal and mudrA TIRs as well as somatic activity and high levels of mudrA expression. Thus, mudrA silencing can be progressively and heritably reversed. Conversely, mudrB expression is never restored, its TIR remains methylated, and new insertions of Mu elements are not observed. These data suggest that mudrA and mudrB silencing may be maintained via distinct mechanisms.

With more than 260,000 species, the angiosperms are the most diverse group of land plants on earth today. Many would argue that their striking diversity stems from the acquisition of the flower along this evolutionary lineage. The argument goes that by enclosing the plant's sex organs, especially the ovule, the flower provided angiosperms with special means to withstand a wide range of environmental conditions, while facilitating pollination or pollinator attraction and seed protection and dispersal. Regardless, the diversity of shapes, colors, and sizes of flowers across the angiosperms is irrefutable and fascinating. Understanding the mechanisms that underlie flower diversity leads us to the understanding, at least in part, of how evolutionary processes have enabled the origin of different forms in nature.

Although the Modern Synthesis has provided a solid framework for understanding how genes evolve in populations, it lacks a theory to satisfactorily explain the evolution of morphological diversity, as it largely marginalized the role of development in the evolution of biological form. Recently, however, an increasing attempt to understand the interrelationships between evolution and development has emerged as a new research field known as evolutionary developmental biology, or, for short, evo-devo. The study of genes involved in different developmental processes, and how changes in these genes or on their regulation can lead to changes in organismal form has become an insightful field.

This dissertation focuses on the evolution and diversification of floral morphology in the Zingiberales and their implications for our understanding of the evolution of plant bauplan. The tropical monocot order Zingiberales provides an excellent framework for evolutionary developmental studies, as changes in floral form throughout the evolution of this group are mainly due to changes of form and function in the petal and stamen whorls, where stamens become infertile and petaloid.

The first part of this dissertation describes how changes in classical floral organ identity genes result in changes in floral organogenesis throughout the evolution of the Zingiberales. First, through a combination of careful morphological studies and genetic approaches, I establish the homology of floral organs, particularly the nature of the so-called `petaloid appendages' on fertile stamens of the ginger group. Second, I show that positive selection is acting upon the AGAMOUS (AG) lineage, and changes in the AG protein suggest a mechanism capable of explaining the morphological changes observed in the Zingiberales flowers.

The latter part of this dissertation goes beyond organ identity genes to investigate the development of organ morphology. In this section, I demonstrate the involvement of the abaxial-adaxial (ab-ad) polarity gene network on the evolution of filament morphology, not only within the Zingiberales but also across all angiosperms, and provide evidence that morphogenetic processes, not just organ identity per se, are driving the evolution of floral form across the order. By studying ab-ad polarity genes, well-known for the establishment of abaxial and adaxial surfaces of leaves, sepals, and petals, I show how the same gene regulatory network has been co-opted during the evolution of angiosperms to shape filament morphology in flowering plants.

I conclude this dissertation by discussing the implications of these findings to our understanding of the mechanisms of plant bauplan evolution. Lastly, I analyze the floral evo-devo research program through a historical and philosophical perspective, hoping to shed light on future directions of research in the field of plant evo-devo, as a consequence of important conceptual changes that this field has undergone in the past two decades.

The microbial world is integrally involved with major ecological processes and affects all domains of life. With the numerous societal challenges we face today, efforts are being directed towards better utilizing microbial communities to support host health and resilience. In regards to agriculture, this includes the fortification of crop production through microbial amendments and microbiome manipulation. To better engineer microbiomes capable of promoting plant growth and ameliorating stress, additional research is needed to untangle the relative contributions of environmental and host factors in recruiting and maintaining beneficial microbes, while repelling potential pathogens. The work presented here seeks to identify and explore the importance of a set of these environmental and host forces in shaping the root and rhizosphere microbiomes of crop species. Specifically, the impacts of disturbance on plant-microbe interactions are characterized in relation to the farming practices of tillage and cover-cropping, heat and drought stress, and host evolution.

This research first explores how a set of widely employed agricultural soil management practices influence the belowground interactions between sorghum, bacteria, and fungi. Currently, it is not well characterized how cultivation systems influence microbiome assembly and activity. Utilizing next generation sequencing methods, we characterized a field system managed for close to two decades with standard tillage or no till practices in combination with either cover-cropping or letting the field lay fallow. We observed a promotion of microbial diversity by standard till and determined that fungal communities responded to a greater degree - in both composition and activity - to management practice than bacteria. Interestingly, despite distinct communities under each regime, similar plant growth outcomes were observed. This work informs understandings of how intermittent soil disturbance impacts agroecosystems and highlights the importance of cross-kingdom analyses.

This work then investigates the combined and isolated impacts of heat and drought stress on sorghum microbiome assembly. Recent studies of drought and the plant microbiome have shown a high degree of variability in bacterial enrichment under drought, particularly for the phylum Actinobacteria. As heat often co-occurs with drought in the field, we sought to determine the relative contributions of temperature to this enrichment. Using a set of controlled growth chamber experiments, we observed that high temperatures do indeed correlate to a restructuring of sorghum-associated bacterial communities. This community differed from what was observed under drought alone, and the majority of indicator taxa within Actinobacteria were not shared between stresses. These results further our knowledge on how different abiotic stresses help modulate community interactions and lay the foundation for additional work characterizing the mechanisms involved in differential microbial enrichment.

In the final chapter of this work, the influence of host evolution on the associations between plants and their belowground microbiome is explored. Past research has shown that plant domestication and polyploidy can broadly influence plant biotic and abiotic interactions. We utilized three approaches - two field studies and one greenhouse-based experiment - to determine whether patterns in bacterial community assembly in wheat roots and rhizospheres could be partially attributable to these host factors. Collectively, we found little evidence of ploidy level and domestication status correlating with shifts in wheat bacterial communities. However, the greatest influence of the host on the microbiome appeared to occur in the rhizosphere compartment, and we suggest future work focuses on this environment to further characterize how host genomic and phenotypic changes influence plant-microbe communications. This research informs perspectives on what key driving forces may underlie microbiome structuring, as well as where future efforts may be best directed towards fortifying plant growth by microbial means.

Taken together, this work addresses fundamental gaps in our knowledge of the plant microbiome and the factors that help govern its structure and function. It demonstrates the ecological importance of agricultural soil management practices on plant-microbial interactions, uncovers the distinct roles of heat and drought stress in plant microbiome assembly, and indicates that domestication and polyploidy are minor contributors in shaping the wheat bacterial microbiome.