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.

Boundary domain genes were recruited to suppress bract growth and promote branching in maize.

Grass inflorescence development is diverse and complex and involves sophisticated but poorly understood interactions of genes regulating branch determinacy and leaf growth. Here, we use a combination of transcript profiling and genetic and phylogenetic analyses to investigate tasselsheath1 (tsh1) and tsh4, two maize genes that simultaneously suppress inflorescence leaf growth and promote branching. We identify a regulatory network of inflorescence leaf suppression that involves the phase change gene tsh4 upstream of tsh1 and the ligule identity gene liguleless2 (lg2). We also find that a series of duplications in the tsh1 gene lineage facilitated its shift from boundary domain in nongrasses to suppressed inflorescence leaves of grasses. Collectively, these results suggest that the boundary domain genes tsh1 and lg2 were recruited to inflorescence leaves where they suppress growth and regulate a nonautonomous signaling center that promotes inflorescence branching, an important component of yield in cereal grasses.

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.

Recruitment of an ancient branching program to suppress carpel development in maize flowers.

Carpels in maize undergo programmed cell death in half of the flowers initiated in ears and in all flowers in tassels. The HD-ZIP I transcription factor gene GRASSY TILLERS1 (GT1) is one of only a few genes known to regulate this process. To identify additional regulators of carpel suppression, we performed a gt1 enhancer screen and found a genetic interaction between gt1 and ramosa3 (ra3). RA3 is a classic inflorescence meristem determinacy gene that encodes a trehalose-6-phosphate (T6P) phosphatase (TPP). Dissection of floral development revealed that ra3 single mutants have partially derepressed carpels, whereas gt1;ra3 double mutants have completely derepressed carpels. Surprisingly, gt1 suppresses ra3 inflorescence branching, revealing a role for gt1 in meristem determinacy. Supporting these genetic interactions, GT1 and RA3 proteins colocalize to carpel nuclei in developing flowers. Global expression profiling revealed common genes misregulated in single and double mutant flowers, as well as in derepressed gt1 axillary meristems. Indeed, we found that ra3 enhances gt1 vegetative branching, similar to the roles for the trehalose pathway and GT1 homologs in the eudicots. This functional conservation over ∼160 million years of evolution reveals ancient roles for GT1-like genes and the trehalose pathway in regulating axillary meristem suppression, later recruited to mediate carpel suppression. Our findings expose hidden pleiotropy of classic maize genes and show how an ancient developmental program was redeployed to sculpt floral form.

Integration of high-density genetic mapping with transcriptome analysis uncovers numerous agronomic QTL and reveals candidate genes for the control of tillering in sorghum.

Phenotypes such as branching, photoperiod sensitivity, and height were modified during plant domestication and crop improvement. Here, we perform quantitative trait locus (QTL) mapping of these and other agronomic traits in a recombinant inbred line (RIL) population derived from an interspecific cross between Sorghum propinquum and Sorghum bicolor inbred Tx7000. Using low-coverage Illumina sequencing and a bin-mapping approach, we generated ∼1920 bin markers spanning ∼875 cM. Phenotyping data were collected and analyzed from two field locations and one greenhouse experiment for six agronomic traits, thereby identifying a total of 30 QTL. Many of these QTL were penetrant across environments and co-mapped with major QTL identified in other studies. Other QTL uncovered new genomic regions associated with these traits, and some of these were environment-specific in their action. To further dissect the genetic underpinnings of tillering, we complemented QTL analysis with transcriptomics, identifying 6189 genes that were differentially expressed during tiller bud elongation. We identified genes such as Dormancy Associated Protein 1 (DRM1) in addition to various transcription factors that are differentially expressed in comparisons of dormant to elongating tiller buds and lie within tillering QTL, suggesting that these genes are key regulators of tiller elongation in sorghum. Our study demonstrates the usefulness of this RIL population in detecting domestication and improvement-associated genes in sorghum, thus providing a valuable resource for genetic investigation and improvement to the sorghum community.

Evolutionary Variation in MADS Box Dimerization Affects Floral Development and Protein Abundance in Maize.

Interactions between MADS box transcription factors are critical in the regulation of floral development, and shifting MADS box protein-protein interactions are predicted to have influenced floral evolution. However, precisely how evolutionary variation in protein-protein interactions affects MADS box protein function remains unknown. To assess the impact of changing MADS box protein-protein interactions on transcription factor function, we turned to the grasses, where interactions between B-class MADS box proteins vary. We tested the functional consequences of this evolutionary variability using maize (Zea mays) as an experimental system. We found that differential B-class dimerization was associated with subtle, quantitative differences in stamen shape. In contrast, differential dimerization resulted in large-scale changes to downstream gene expression. Differential dimerization also affected B-class complex composition and abundance, independent of transcript levels. This indicates that differential B-class dimerization affects protein degradation, revealing an important consequence for evolutionary variability in MADS box interactions. Our results highlight complexity in the evolution of developmental gene networks: changing protein-protein interactions could affect not only the composition of transcription factor complexes but also their degradation and persistence in developing flowers. Our results also show how coding change in a pleiotropic master regulator could have small, quantitative effects on development.

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.

The regulatory landscape of a core maize domestication module controlling bud dormancy and growth repression.

Many domesticated crop plants have been bred for increased apical dominance, displaying greatly reduced axillary branching compared to their wild ancestors. In maize, this was achieved through selection for a gain-of-function allele of the TCP transcription factor teosinte branched1 (tb1). The mechanism for how a dominant Tb1 allele increased apical dominance, is unknown. Through ChIP seq, RNA seq, hormone and sugar measurements on 1 mm axillary bud tissue, we identify the genetic pathways putatively regulated by TB1. These include pathways regulating phytohormones such as gibberellins, abscisic acid and jasmonic acid, but surprisingly, not auxin. In addition, metabolites involved in sugar sensing such as trehalose 6-phosphate were increased. This suggests that TB1 induces bud suppression through the production of inhibitory phytohormones and by reducing sugar levels and energy balance. Interestingly, TB1 also putatively targets several other domestication loci, including teosinte glume architecture1, prol1.1/grassy tillers1, as well as itself. This places tb1 on top of the domestication hierarchy, demonstrating its critical importance during the domestication of maize from teosinte.

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 meristem determinacy.

The grass inflorescence is striking not only for its beauty and diversity, but also for its developmental complexity. While models of inflorescence architecture have been proposed in both eudicots and grasses, these are inadequate to fully explain the complex branching events that occur during the development of the grass inflorescence. Key to understanding grass inflorescence architecture is the meristem determinacy/indeterminacy decision, which regulates the number of branching events that occur. Here we review what has been learned about meristem determinacy from grass mutants with defects in inflorescence development. A picture is emerging of a complex network of signaling molecules and meristem identity factors that interact to regulate inflorescence meristem activity, many of which have been modified during crop domestication directly affecting yield traits.

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.

Altered expression of maize PLASTOCHRON1 enhances biomass and seed yield by extending cell division duration.

Maize is the highest yielding cereal crop grown worldwide for grain or silage. Here, we show that modulating the expression of the maize PLASTOCHRON1 (ZmPLA1) gene, encoding a cytochrome P450 (CYP78A1), results in increased organ growth, seedling vigour, stover biomass and seed yield. The engineered trait is robust as it improves yield in an inbred as well as in a panel of hybrids, at several locations and over multiple seasons in the field. Transcriptome studies, hormone measurements and the expression of the auxin responsive DR5rev:mRFPer marker suggest that PLA1 may function through an increase in auxin. Detailed analysis of growth over time demonstrates that PLA1 stimulates the duration of leaf elongation by maintaining dividing cells in a proliferative, undifferentiated state for a longer period of time. The prolonged duration of growth also compensates for growth rate reduction caused by abiotic stresses.

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.

Evolutionary Dynamics of Floral Homeotic Transcription Factor Protein-Protein Interactions.

Protein-protein interactions (PPIs) have widely acknowledged roles in the regulation of development, but few studies have addressed the timing and mechanism of shifting PPIs over evolutionary history. The B-class MADS-box transcription factors, PISTILLATA (PI) and APETALA3 (AP3) are key regulators of floral development. PI-like (PI(L)) and AP3-like (AP3(L)) proteins from a number of plants, including Arabidopsis thaliana (Arabidopsis) and the grass Zea mays (maize), bind DNA as obligate heterodimers. However, a PI(L) protein from the grass relative Joinvillea can bind DNA as a homodimer. To ascertain whether Joinvillea PI(L) homodimerization is an anomaly or indicative of broader trends, we characterized PI(L) dimerization across the Poales and uncovered unexpected evolutionary lability. Both obligate B-class heterodimerization and PI(L) homodimerization have evolved multiple times in the order, by distinct molecular mechanisms. For example, obligate B-class heterodimerization in maize evolved very recently from PI(L) homodimerization. A single amino acid change, fixed during domestication, is sufficient to toggle one maize PI(L) protein between homodimerization and obligate heterodimerization. We detected a signature of positive selection acting on residues preferentially clustered in predicted sites of contact between MADS-box monomers and dimers, and in motifs that mediate MADS PPI specificity in Arabidopsis. Changing one positively selected residue can alter PI(L) dimerization activity. Furthermore, ectopic expression of a Joinvillea PI(L) homodimer in Arabidopsis can homeotically transform sepals into petals. Our results provide a window into the evolutionary remodeling of PPIs, and show that novel interactions have the potential to alter plant form in a context-dependent manner.

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.