Ana Maria Rocha de Almeida

The banana (Musa acuminata) genome and the evolution of monocotyledonous plants.

Bananas (Musa spp.), including dessert and cooking types, are giant perennial monocotyledonous herbs of the order Zingiberales, a sister group to the well-studied Poales, which include cereals. Bananas are vital for food security in many tropical and subtropical countries and the most popular fruit in industrialized countries. The Musa domestication process started some 7,000 years ago in Southeast Asia. It involved hybridizations between diverse species and subspecies, fostered by human migrations, and selection of diploid and triploid seedless, parthenocarpic hybrids thereafter widely dispersed by vegetative propagation. Half of the current production relies on somaclones derived from a single triploid genotype (Cavendish). Pests and diseases have gradually become adapted, representing an imminent danger for global banana production. Here we describe the draft sequence of the 523-megabase genome of a Musa acuminata doubled-haploid genotype, providing a crucial stepping-stone for genetic improvement of banana. We detected three rounds of whole-genome duplications in the Musa lineage, independently of those previously described in the Poales lineage and the one we detected in the Arecales lineage. This first monocotyledon high-continuity whole-genome sequence reported outside Poales represents an essential bridge for comparative genome analysis in plants. As such, it clarifies commelinid-monocotyledon phylogenetic relationships, reveals Poaceae-specific features and has led to the discovery of conserved non-coding sequences predating monocotyledon–eudicotyledon divergence.

Two Evolutionarily Distinct Classes of Paleopolyploidy

Whole genome duplications (WGDs) occurred in the distant evolutionary history of many lineages and are particularly frequent in the flowering plant lineages. Following paleopolyploidization in plants, most duplicated genes are deleted by intrachromosomal recombination, a process referred to as fractionation. In the examples studied so far, genes are disproportionately lost from one of the parental subgenomes (biased fractionation) and the subgenome having lost the lowest number of genes is more expressed (genome dominance). In the present study, we analyzed the pattern of gene deletion and gene expression following the most recent WGD in banana (alpha event) and extended our analyses to seven other sequenced plant genomes: poplar, soybean, medicago, arabidopsis, sorghum, brassica, and maize. We propose a new class of ancient WGD, with Musa (alpha), poplar, and soybean as members, where genes are both deleted and expressed to an equal extent (unbiased fractionation and genome equivalence). We suggest that WGDs with genome dominance and biased fractionation (Class I) may result from ancient allotetraploidies, while WGDs without genome dominance or biased fractionation (Class II) may result from ancient autotetraploidies.

Molecular evolution and patterns of duplication in the SEP/AGL6-like lineage of the Zingiberales: a proposed mechanism for floral diversification.

The diversity of floral forms in the plant order Zingiberales has evolved through alterations in floral organ morphology. One striking alteration is the shift from fertile, filamentous stamens to sterile, laminar (petaloid) organs in the stamen whorls, attributed to specific pollination syndromes. Here, we examine the role of the SEPALLATA (SEP) genes, known to be important in regulatory networks underlying floral development and organ identity, in the evolution of development of the diverse floral organs phenotypes in the Zingiberales. Phylogenetic analyses show that the SEP-like genes have undergone several duplication events giving rise to multiple copies. Selection tests on the SEP-like genes indicate that the two copies of SEP3 have mostly evolved under balancing selection, probably due to strong functional restrictions as a result of their critical role in floral organ specification. In contrast, the two LOFSEP copies have undergone differential positive selection, indicating neofunctionalization. Reverse transcriptase-polymerase chain reaction, gene expression from RNA-seq data, and in situ hybridization analyses show that the recovered genes have differential expression patterns across the various whorls and organ types found in the Zingiberales. Our data also suggest that AGL6, sister to the SEP-like genes, may play an important role in stamen morphology in the Zingiberales. Thus, the SEP-like genes are likely to be involved in some of the unique morphogenetic patterns of floral organ development found among this diverse order of tropical monocots. This work contributes to a growing body of knowledge focused on understanding the role of gene duplications and the evolution of entire gene networks in the evolution of flower development.

A Method for Extracting High-Quality RNA from Diverse Plants for Next-Generation Sequencing and Gene Expression Analyses

Premise of the study: To study gene expression in plants, high-quality RNA must be extracted in quantities sufficient for subsequent cDNA library construction. Field-based collections are often limited in quantity and quality of tissue and are typically preserved in RNAlater. Obtaining sufficient and high-quality yield from variously preserved samples is essential to studies of comparative biology. We present a protocol for the extraction of high-quality RNA from even the most recalcitrant plant tissues. Methods and Results: Tissues from mosses, cycads, and angiosperm floral organs and leaves were preserved in RNAlater or frozen fresh at −80°C. Extractions were performed and quality was measured for yield and purity. Conclusions: This protocol results in the extraction of high-quality RNA from a variety of plant tissues representing vascular and nonvascular plants. RNA was used for cDNA synthesis to generate libraries for next-generation sequencing and for expression studies using quantitative PCR (qPCR) and semiquantitative reverse transcription PCR (RT-PCR).

Tracking the development of the petaloid fertile stamen in Canna indica: insights into the origin of androecial petaloidy in the Zingiberales

The order Zingiberales comprises ∼2500 species of tropical to subtropical plants, including agriculturally (e.g. banana, ginger) and horticulturally (e.g. cannas, heliconias, bird-of-paradise) important plants. Throughout the evolution of this order, the stamens have been modified from the ancestral filamentous structures that produce pollen (seen in Banana flowers) to petal-like structures that no longer bear pollen sacs (seen in Canna flowers). This results in a reduction of pollen, but an effective increase in the overall size of the floral display and perhaps in the efficacy of specialized pollinators by converting stamens into ‘petals’. This study investigates the genetic mechanisms that are involved in making petal-like structures in place of pollen-producing stamens.

Homoplasy, Pollination, and Emerging Complexity During the Evolution of Floral Development in the Tropical Gingers (Zingiberales)

With their impressive array of floral diversity and a largely-understood phylogenetic relationships, the Zingiberales provide an ideal model clade to test for the roles of genetic and ecological factors driving floral diversification. Many Zingiberales have close associations with particular suites of pollinators, a species-level interaction that is reflected in their overall floral morphology. Here we first discuss the importance of understanding developmental evolution in a phylogenetic context, then use the evolution of floral morphology across the Zingiberales to test the hypothesis that shifts in rates of diversification among these tropical monocots is correlated with shifts in pollination syndrome, suggesting an important role of pollination specificity in driving speciation and floral diversification in the Zingiberales.

Positive selection on the K domain of the AGAMOUS protein in the Zingiberales suggests a mechanism for the evolution of androecial morphology

BackgroundThe ABC model of flower development describes the molecular basis for specification of floral organ identity in model eudicots such as Arabidopsis and Antirrhinum. According to this model, expression of C-class genes is linked to stamen and gynoecium organ identity. The Zingiberales is an order of tropical monocots in which the evolution of floral morphology is characterized by a marked increase in petaloidy in the androecium. Petaloidy is a derived characteristic of the ginger families and seems to have arisen in the common ancestor of the ginger clade. We hypothesize that duplication of the C-class AGAMOUS (AG) gene followed by divergence of the duplicated AG copies during the diversification of the ginger clade lineages explains the evolution of petaloidy in the androecium. In order to address this hypothesis, we carried out phylogenetic analyses of the AG gene family across the Zingiberales and investigated patterns of gene expression within the androecium.ResultsPhylogenetic analysis supports a scenario in which Zingiberales-specific AG genes have undergone at least one round of duplication. Gene duplication was immediately followed by divergence of the retained copies. In particular, we detect positive selection in the third alpha-helix of the K domain of Zingiberales AGAMOUS copy 1 (ZinAG-1). A single fixed amino acid change is observed in ZinAG-1 within the ginger clade when compared to the banana grade. Expression analyses of AG and APETALA1/FRUITFULL (AP1/FUL) in Musa basjoo is similar to A- and C-class gene expressions in the Arabidopsis thaliana model, while Costus spicatus exhibits simultaneous expression of AG and AP1/FUL in most floral organs. We propose that this novel expression pattern could be correlated with the evolution of androecial petaloidy within the Zingiberales.ConclusionsOur results present an intricate story in which duplication of the AG lineage has lead to the retention of at least two diverged Zingiberales-specific copies, ZinAG-1 and Zingiberales AGAMOUS copy 2 (ZinAG-2). Positive selection on ZinAG-1 residues suggests a mechanism by which AG gene divergence may explain observed morphological changes in Zingiberales flowers. Expression data provides preliminary support for the proposed mechanism, although further studies are required to fully test this hypothesis.

Evolution of petaloidy in the zingiberales: An assessment of the relationship between ultrastructure and gene expression patterns

Background: The development of petal‐like organs has occurred repetitively throughout angiosperm evolution. Despite homoplasy, it is possible that common underlying molecular mechanisms are repeatedly recruited to drive the development of petaloid organs. In Zingiberales, infertile, petal‐like structures replace fertile stamens, resulting in petaloidy in androecial whorls. Assuming that androecial petaloidy is a shared derived characteristic, we expect to find common ultrastructure and molecular mechanisms underlying androecial petaloidy across Zingiberales. Results: We show that petaloidy in Zingiberales is associated with tightly packed, protruding epidermal cells. Expression patterns for candidate genes involved in petal identity differ between the petaloid organs of Costaceae v. Cannaceae, despite similar macro‐ and microscopic organization. For all candidate gene families analyzed, our data suggest at least one Zingiberales‐specific duplication event. Conclusions: Our data suggest that the patterns of B‐class gene expression across the Zingiberales do not correlate with the occurrence of petaloidy, indicating that androecial petaloidy might have evolved independently of B‐class gene expression in some lineages. It is possible that gene duplication may play a role in the diversity of petaloid structures found throughout the Zingiberales. It is likely that Zingiberales petaloidy may also result from the deployment of genes other than those involved in specification of petal identity. Developmental Dynamics 244:1121–1132, 2015.

Co-option of the polarity gene network shapes filament morphology in angiosperms

The molecular genetic mechanisms underlying abaxial-adaxial polarity in plants have been studied as a property of lateral and flattened organs, such as leaves. In leaves, laminar expansion occurs as a result of balanced abaxial-adaxial gene expression. Over- or under- expression of either abaxializing or adaxializing genes inhibits laminar growth, resulting in a mutant radialized phenotype. Here, we show that co-option of the abaxial-adaxial polarity gene network plays a role in the evolution of stamen filament morphology in angiosperms. RNA-Seq data from species bearing laminar (flattened) or radial (cylindrical) filaments demonstrates that species with laminar filaments exhibit balanced expression of abaxial-adaxial (ab-ad) genes, while overexpression of a YABBY gene is found in species with radial filaments. This result suggests that unbalanced expression of ab-ad genes results in inhibition of laminar outgrowth, leading to a radially symmetric structure as found in many angiosperm filaments. We anticipate that co-option of the polarity gene network is a fundamental mechanism shaping many aspects of plant morphology during angiosperm evolution.

Loss of YABBY2-Like Gene Expression May Underlie the Evolution of the Laminar Style in Canna and Contribute to Floral Morphological Diversity in the Zingiberales

The Zingiberales is an order of tropical monocots that exhibits diverse floral morphologies. The evolution of petaloid, laminar stamens, staminodes, and styles contributes to this diversity. The laminar style is a derived trait in the family Cannaceae and plays an important role in pollination as its surface is used for secondary pollen presentation. Previous work in the Zingiberales has implicated YABBY2-like genes, which function in promoting laminar outgrowth, in the evolution of stamen morphology. Here, we investigate the evolution and expression of Zingiberales YABBY2-like genes in order to understand the evolution of the laminar style in Canna. Phylogenetic analyses show that multiple duplication events have occurred in this gene lineage prior to the diversification of the Zingiberales. Reverse transcription-PCR in Canna, Costus, and Musa reveals differential expression across floral organs, taxa, and gene copies, and a role for YABBY2-like genes in the evolution of the laminar style is proposed. Selection tests indicate that almost all sites in conserved domains are under purifying selection, consistent with their functional relevance, and a motif unique to monocot YABBY2-like genes is identified. These results contribute to our understanding of the molecular mechanisms underlying the evolution of floral morphologies.