QTL analysis of divergent floral morphology traits between Gilia yorkii and G. capitata.

Speciation is a complex process typically accompanied by significant genetic and morphological differences between sister populations. In plants, divergent floral morphologies and pollinator differences can result in reproductive isolation between populations. Here, we explore floral trait differences between two recently diverged species, Gilia yorkii and G. capitata. The distributions of floral traits in parental, F1, and F2 populations are compared, and groups of correlated traits are identified. We describe the genetic architecture of floral traits through a quantitative trait locus (QTL) analysis using an F2 population of 187 individuals. While all identified QTLs were of moderate (10-25%) effect, interestingly, most QTL intervals were non-overlapping, suggesting that, in general, traits do not share a common genetic basis. Our results provide a framework for future identification of genes involved in the evolution of floral morphology.

Ontogeny and anatomy of Bouteloua (Poaceae: Chloridoideae) species display a basipetal branch formation and a novel modified leaf structure in grasses.

BACKGROUND AND AIMS: Shoot ontogenesis in grasses follows a transition from a vegetative phase into a reproductive phase. Current studies provide insight into how branch and spikelet formation occur during the reproductive phase. However, these studies do not explain all the complex diversity of grass inflorescence forms and are mostly focused on model grasses. Moreover, truncated inflorescences of the non-model grass genus Urochloa (Panicoideae) with formation of primary branches have basipetal initiation of branches. Bouteloua species (Chloridoideae) are non-model grasses that form truncated inflorescences of primary branches with apical vestiges of uncertain homology at the tips of branching events and sterile florets above the lowermost fertile floret. Sterile florets are reduced to rudimentary lemmas composed of three large awns diverging from an awn column. Conflict about the awn column identity of this rudimentary lemma is often addressed in species descriptions of this genus. We test if Bouteloua species can display basipetal initiation of branches and explore the identity of vestiges and the awn column of rudimentary lemmas. METHODS: We surveyed the inflorescence ontogeny and branch/awn anatomy of Bouteloua species and compared results with recent ontogenetic studies of chloridoids. KEY RESULTS: Bouteloua arizonica has florets with basipetal maturation. Branches display basipetal branch initiation and maturation. Branch vestiges are formed laterally by meristems during early branching events. The spikelet meristem forms the awn column of rudimentary lemmas. Vestiges and sterile floret awns have anatomical similarities to C4 leaves. CONCLUSIONS: Basipetal initiation of branches is a novel feature for Chloridoideae grasses. Branch vestiges are novel vegetative grass structures. Sterile floret awn columns are likely to be extensions of the rachilla.

Chromosome-Scale Genome Assembly of Gilia yorkii Enables Genetic Mapping of Floral Traits in an Interspecies Cross.

Substantial morphological variation in land plants remains inaccessible to genetic analysis because current models lack variation in important ecological and agronomic traits. The genus Gilia was historically a model for biosystematics studies and includes variation in morphological traits that are poorly understood at the genetic level. We assembled a chromosome-scale reference genome of G. yorkii and used it to investigate genome evolution in the Polemoniaceae. We performed QTL (quantitative trait loci) mapping in a G. yorkii×G. capitata interspecific population for traits related to inflorescence architecture and flower color. The genome assembly spans 2.75 Gb of the estimated 2.80-Gb genome, with 96.7% of the sequence contained in the nine largest chromosome-scale scaffolds matching the haploid chromosome number. Gilia yorkii experienced at least one round of whole-genome duplication shared with other Polemoniaceae after the eudicot paleohexaploidization event. We identified QTL linked to variation in inflorescence architecture and petal color, including a candidate for the major flower color QTL-a tandem duplication of flavanol 3',5'-hydroxylase. Our results demonstrate the utility of Gilia as a forward genetic model for dissecting the evolution of development in plants including the causal loci underlying inflorescence architecture transitions.

Development of an Evolutionary Tree Concept Inventory.

Despite the importance of tree-thinking and evolutionary trees to biology, no appropriately developed concept inventory exists to measure student understanding of these important concepts. To address this need, we developed a multiple-choice concept inventory consisting of 24 pairs of items, and we provide evidence to support its use among undergraduate students. A set of learning outcomes was developed to guide the creation of the concept inventory. The learning outcomes, student interviews, and student responses were used to develop and revise inventory items. Supporting evidence was gathered from traditional item analysis, exploratory factor analysis, confirmatory factor analysis, traditional reliability analyses, and comparisons to alternative assessments. Appropriate implementation and utility of the concept inventory are discussed.

Bulked-Segregant Analysis Coupled to Whole Genome Sequencing (BSA-Seq) for Rapid Gene Cloning in Maize.

Forward genetics remains a powerful method for revealing the genes underpinning organismal form and function, and for revealing how these genes are tied together in gene networks. In maize, forward genetics has been tremendously successful, but the size and complexity of the maize genome made identifying mutant genes an often arduous process with traditional methods. The next generation sequencing revolution has allowed for the gene cloning process to be significantly accelerated in many organisms, even when genomes are large and complex. Here, we describe a bulked-segregant analysis sequencing (BSA-Seq) protocol for cloning mutant genes in maize. Our simple strategy can be used to quickly identify a mapping interval and candidate single nucleotide polymorphisms (SNPs) from whole genome sequencing of pooled F2 individuals. We employed this strategy to identify narrow odd dwarf as an enhancer of teosinte branched1, and to identify a new allele of defective kernel1 Our method provides a quick, simple way to clone genes in maize.

Grass inflorescence architecture and evolution: the origin of novel signaling centers.

Contents 367 I. 367 II. 368 III. 370 IV. 371 371 References 371 SUMMARY: A central goal of evo-devo is to understand how morphological diversity arises from existing developmental mechanisms, requiring a clear, predictive explanatory framework of the underlying developmental mechanisms. Despite an ever-increasing literature on genes regulating grass inflorescence development, an effective model of inflorescence patterning is lacking. I argue that the existing framework for grass inflorescence development, which invokes homeotic shifts in multiple distinct meristem identities, obscures a recurring theme emerging from developmental genetic studies in grass models, that is that inflorescence branching is regulated by novel localized signaling centers. Understanding the origin and function of these novel signaling centers will be key to future evo-devo work on the grass inflorescence.

A Gene for Genetic Background in Zea mays: Fine-Mapping enhancer of teosinte branched1.2 to a YABBY Class Transcription Factor.

The effects of an allelic substitution at a gene often depend critically on genetic background, i.e., the genotypes at other genes in the genome. During the domestication of maize from its wild ancestor (teosinte), an allelic substitution at teosinte branched (tb1) caused changes in both plant and ear architecture. The effects of tb1 on phenotype were shown to depend on multiple background loci, including one called enhancer of tb1.2 (etb1.2). We mapped etb1.2 to a YABBY class transcription factor (ZmYAB2.1) and showed that the maize alleles of ZmYAB2.1 are either expressed at a lower level than teosinte alleles or disrupted by insertions in the sequences. tb1 and etb1.2 interact epistatically to control the length of internodes within the maize ear, which affects how densely the kernels are packed on the ear. The interaction effect is also observed at the level of gene expression, with tb1 acting as a repressor of ZmYAB2.1 expression. Curiously, ZmYAB2.1 was previously identified as a candidate gene for another domestication trait in maize, nonshattering ears. Consistent with this proposed role, ZmYAB2.1 is expressed in a narrow band of cells in immature ears that appears to represent a vestigial abscission (shattering) zone. Expression in this band of cells may also underlie the effect on internode elongation. The identification of ZmYAB2.1 as a background factor interacting with tb1 is a first step toward a gene-level understanding of how tb1 and the background within which it works evolved in concert during maize domestication.

Prevalence and Persistence of Misconceptions in Tree Thinking.

Darwin described evolution as "descent with modification." Descent, however, is not an explicit focus of most evolution instruction and often leaves deeply held misconceptions to dominate student understanding of common ancestry and species relatedness. Evolutionary trees are ways of visually depicting descent by illustrating the relationships between species and groups of species. The ability to properly interpret and use evolutionary trees has become known as "tree thinking." We used a 20-question assessment to measure misconceptions in tree thinking and compare the proportion of students who hold these misconceptions in an introductory biology course with students in two higher-level courses including a senior level biology course. We found that misconceptions related to reading the graphic (reading the tips and node counting) were variably influenced across time with reading the tips decreasing and node counting increasing in prevalence. On the other hand, misconceptions related to the fundamental underpinnings of evolutionary theory (ladder thinking and similarity equals relatedness) proved resistant to change during a typical undergraduate study of biology. A possible new misconception relating to the length of the branches in an evolutionary tree is described. Understanding the prevalence and persistence of misconceptions informs educators as to which misconceptions should be targeted in their courses.

The Maize PI/GLO Ortholog Zmm16/sterile tassel silky ear1 Interacts with the Zygomorphy and Sex Determination Pathways in Flower Development.

In monocots and eudicots, B class function specifies second and third whorl floral organ identity as described in the classic ABCE model. Grass B class APETALA3/DEFICIENS orthologs have been functionally characterized; here, we describe the positional cloning and characterization of a maize (Zea mays) PISTILLATA/GLOBOSA ortholog Zea mays mads16 (Zmm16)/sterile tassel silky ear1 (sts1). We show that, similar to many eudicots, all the maize B class proteins bind DNA as obligate heterodimers and positively regulate their own expression. However, sts1 mutants have novel phenotypes that provide insight into two derived aspects of maize flower development: carpel abortion and floral asymmetry. Specifically, we show that carpel abortion acts downstream of organ identity and requires the growth-promoting factor grassy tillers1 and that the maize B class genes are expressed asymmetrically, likely in response to zygomorphy of grass floral primordia. Further investigation reveals that floral phyllotactic patterning is also zygomorphic, suggesting significant mechanistic differences with the well-characterized models of floral polarity. These unexpected results show that despite extensive study of B class gene functions in diverse flowering plants, novel insights can be gained from careful investigation of homeotic mutants outside the core eudicot model species.

Positional cloning in maize (Zea mays subsp. mays, Poaceae).

PREMISE OF THE STUDY: Positional (or map-based) cloning is a common approach to identify the molecular lesions causing mutant phenotypes. Despite its large and complex genome, positional cloning has been recently shown to be feasible in maize, opening up a diverse collection of mutants to molecular characterization. • METHODS AND RESULTS: Here we outline a general protocol for positional cloning in maize. While the general strategy is similar to that used in other plant species, we focus on the unique resources and approaches that should be considered when applied to maize mutants. • CONCLUSIONS: Positional cloning approaches are appropriate for maize mutants and quantitative traits, opening up to molecular characterization the large array of genetic diversity in this agronomically important species. The cloning approach described should be broadly applicable to other species as more plant genomes become available.

Protein change in plant evolution: tracing one thread connecting molecular and phenotypic diversity.

Proteins change over the course of evolutionary time. New protein-coding genes and gene families emerge and diversify, ultimately affecting an organism's phenotype and interactions with its environment. Here we survey the range of structural protein change observed in plants and review the role these changes have had in the evolution of plant form and function. Verified examples tying evolutionary change in protein structure to phenotypic change remain scarce. We will review the existing examples, as well as draw from investigations into domestication, and quantitative trait locus (QTL) cloning studies searching for the molecular underpinnings of natural variation. The evolutionary significance of many cloned QTL has not been assessed, but all the examples identified so far have begun to reveal the extent of protein structural diversity tolerated in natural systems. This molecular (and phenotypic) diversity could come to represent part of natural selection's source material in the adaptive evolution of novel traits. Protein structure and function can change in many distinct ways, but the changes we identified in studies of natural diversity and protein evolution were predicted to fall primarily into one of six categories: altered active and binding sites; altered protein-protein interactions; altered domain content; altered activity as an activator or repressor; altered protein stability; and hypomorphic and hypermorphic alleles. There was also variability in the evolutionary scale at which particular changes were observed. Some changes were detected at both micro- and macroevolutionary timescales, while others were observed primarily at deep or shallow phylogenetic levels. This variation might be used to determine the trajectory of future investigations in structural molecular evolution.

From many, one: genetic control of prolificacy during maize domestication.

A reduction in number and an increase in size of inflorescences is a common aspect of plant domestication. When maize was domesticated from teosinte, the number and arrangement of ears changed dramatically. Teosinte has long lateral branches that bear multiple small ears at their nodes and tassels at their tips. Maize has much shorter lateral branches that are tipped by a single large ear with no additional ears at the branch nodes. To investigate the genetic basis of this difference in prolificacy (the number of ears on a plant), we performed a genome-wide QTL scan. A large effect QTL for prolificacy (prol1.1) was detected on the short arm of chromosome 1 in a location that has previously been shown to influence multiple domestication traits. We fine-mapped prol1.1 to a 2.7 kb "causative region" upstream of the grassy tillers1 (gt1) gene, which encodes a homeodomain leucine zipper transcription factor. Tissue in situ hybridizations reveal that the maize allele of prol1.1 is associated with up-regulation of gt1 expression in the nodal plexus. Given that maize does not initiate secondary ear buds, the expression of gt1 in the nodal plexus in maize may suppress their initiation. Population genetic analyses indicate positive selection on the maize allele of prol1.1, causing a partial sweep that fixed the maize allele throughout most of domesticated maize. This work shows how a subtle cis-regulatory change in tissue specific gene expression altered plant architecture in a way that improved the harvestability of maize.

Early inflorescence development in the grasses (Poaceae).

The shoot apical meristem of grasses produces the primary branches of the inflorescence, controlling inflorescence architecture and hence seed production. Whereas leaves are produced in a distichous pattern, with the primordia separated from each other by an angle of 180°, inflorescence branches are produced in a spiral in most species. The morphology and developmental genetics of the shift in phyllotaxis have been studied extensively in maize and rice. However, in wheat, Brachypodium, and oats, all in the grass subfamily Pooideae, the change in phyllotaxis does not occur; primary inflorescence branches are produced distichously. It is unknown whether the distichous inflorescence originated at the base of Pooideae, or whether it appeared several times independently. In this study, we show that Brachyelytrum, the genus sister to all other Pooideae has spiral phyllotaxis in the inflorescence, but that in the remaining 3000+ species of Pooideae, the phyllotaxis is two-ranked. These two-ranked inflorescences are not perfectly symmetrical, and have a clear "front" and "back;" this developmental axis has never been described in the literature and it is unclear what establishes its polarity. Strictly distichous inflorescences appear somewhat later in the evolution of the subfamily. Two-ranked inflorescences also appear in a few grass outgroups and sporadically elsewhere in the family, but unlike in Pooideae do not generally correlate with a major radiation of species. After production of branches, the inflorescence meristem may be converted to a spikelet meristem or may simply abort; this developmental decision appears to be independent of the branching pattern.

grassy tillers1 promotes apical dominance in maize and responds to shade signals in the grasses.

The shape of a plant is largely determined by regulation of lateral branching. Branching architecture can vary widely in response to both genotype and environment, suggesting regulation by a complex interaction of autonomous genetic factors and external signals. Tillers, branches initiated at the base of grass plants, are suppressed in response to shade conditions. This suppression of tiller and lateral branch growth is an important trait selected by early agriculturalists during maize domestication and crop improvement. To understand how plants integrate external environmental cues with endogenous signals to control their architecture, we have begun a functional characterization of the maize mutant grassy tillers1 (gt1). We isolated the gt1 gene using positional cloning and found that it encodes a class I homeodomain leucine zipper gene that promotes lateral bud dormancy and suppresses elongation of lateral ear branches. The gt1 expression is induced by shading and is dependent on the activity of teosinte branched1 (tb1), a major domestication locus controlling tillering and lateral branching. Interestingly, like tb1, gt1 maps to a quantitative trait locus that regulates tillering and lateral branching in maize and shows evidence of selection during maize domestication. Branching and shade avoidance are both of critical agronomic importance, but little is known about how these processes are integrated. Our results indicate that gt1 mediates the reduced branching associated with the shade avoidance response in the grasses. Furthermore, selection at the gt1 locus suggests that it was involved in improving plant architecture during the domestication of maize.

A conserved mechanism of bract suppression in the grass family.

Suppression of inflorescence leaf, or bract, growth has evolved multiple times in diverse angiosperm lineages, including the Poaceae and Brassicaceae. Studies of Arabidopsis thaliana mutants have revealed several genes involved in bract suppression, but it is not known if these genes play a similar role in other plants with suppressed bracts. We identified maize (Zea mays) tassel sheath (tsh) mutants, characterized by the loss of bract suppression, that comprise five loci (tsh1-tsh5). We used map-based cloning to identify Tsh1 and found that it encodes a GATA zinc-finger protein, a close homolog of HANABA TARANU (HAN) of Arabidopsis. The bract suppression function of Tsh1 is conserved throughout the grass family, as we demonstrate that the rice (Oryza sativa) NECK LEAF1 (NL1) and barley (Hordeum vulgare) THIRD OUTER GLUME (TRD) genes are orthologous with Tsh1. Interestingly, NL1/Tsh1/TRD expression and function are not conserved with HAN. The existence of paralogous NL1/Tsh1/TRD-like genes in the grasses indicates that the NL1/Tsh1/TRD lineage was created by recent duplications that may have facilitated its neofunctionalization. A comparison with the Arabidopsis genes regulating bract suppression further supports the hypothesis that the convergent evolution of bract suppression in the Poaceae involved recruitment of a distinct genetic pathway.

Conservation of B class gene expression in the second whorl of a basal grass and outgroups links the origin of lodicules and petals.

Studies of flower development in core eudicot species have established a central role for B class MADS-box genes in specifying petal and stamen identities. Similarly in maize and rice, B class genes are essential for lodicule and stamen specification, suggesting homology of petals and lodicules and conservation of B class gene activity across angiosperms. However, lodicules are grass-specific organs with a morphology distinct from petals, thus their true homology to eudicot and nongrass monocot floral organs has been a topic of debate. To understand the relationship of lodicules to the sterile floral organs of nongrass monocots we have isolated and observed the expression of B class genes from a basal grass Streptochaeta that diverged before the evolution of lodicules, as well as the outgroups Joinvillea and Elegia, which have a typical monocot floral plan. Our results support a conserved role for B function genes across the angiosperms and provide additional evidence linking the evolution of lodicules and second whorl tepal/petals of monocots. The expression data and morphological analysis suggest that the function of B class genes should be broadly interpreted as required for differentiation of a distinct second floral whorl as opposed to specifying petal identity per se.

Conservation of B-class floral homeotic gene function between maize and Arabidopsis.

The ABC model of flower development, established through studies in eudicot model species, proposes that petal and stamen identity are under the control of B-class genes. Analysis of B- and C-class genes in the grass species rice and maize suggests that the C- and B-class functions are conserved between monocots and eudicots, with B-class genes controlling stamen and lodicule development. We have undertaken a further analysis of the maize B-class genes Silky1, the putative AP3 ortholog, and Zmm16, a putative PI ortholog, in order to compare their function with the Arabidopsis B-class genes. Our results show that maize B-class proteins interact in vitro to bind DNA as an obligate heterodimer, as do Arabidopsis B-class proteins. The maize proteins also interact with the appropriate Arabidopsis B-class partner proteins to bind DNA. Furthermore, we show that maize B-class genes are capable of rescuing the corresponding Arabidopsis B-class mutant phenotypes. This demonstrates B-class activity of the maize gene Zmm16, and provides compelling evidence that B-class gene function is conserved between monocots and eudicots.