Integrating new variables into a framework to support cacao denomination of origin: a case study in Southwest Colombia.

BACKGROUND: Cocoa quality plays a pivotal role in establishing denominations of origin, with genotypes, geography, climate and soil conditions being key variables. However, these factors have not been comprehensively explored in defining cacao denominations of origin. The present study addresses this gap by laying the foundation for cacao denomination of origin, focusing on the Buenaventura region on Colombia's Pacific coast. Our goal is to provide a holistic understanding of the elements underpinning cacao denomination of origin, emphasizing Buenaventura's unique cocoa quality and geographical significance. RESULTS: Through the Buenaventura case, we propose a robust framework applicable to other cacao-producing regions, elevating the recognition and value of cacao denomination of origin. Our framework encompasses geography, agronomy, genetics, microbial diversity, pests and diseases and cocoa quality. In a pioneering move, we propose a cacao denomination of origin in Colombia, specifically examining Bajo Calima, Sabaletas and Cisneros within Buenaventura region. Buenaventura stands out for its cocoa quality, characterized by fruity flavors attributed to the rich biodiversity of the lowland rainforest. CONCLUSION: Our analysis indicates specific geographical indicators for each of the study zones, with Buenaventura identified as a region with natural characteristics to produce fine flavour cocoa products. Each zone exhibited a high differentiation and diversity of cacao cultivars. Buenaventura has the potential to be designated as a future denomination of origin for cacao from the Pacific region of Colombia, characterized by its unique fruity-aroma chocolates. Our framework is adaptable to other cacao-producing regions, facilitating the establishment of denominations of origin within the cocoa industry and agriculture. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Characterizing subgenome recombination and chromosomal imbalances in banana varietal lineages.

BACKGROUND: Bananas and plantains (Musa spp.) are among the most important crops worldwide. The cultivated varieties are vegetatively propagated, so their genetic diversity is essentially fixed over time. Musa acuminata, M. balbisiana and M. schizocarpa have provided the named A, B and S subgenomes that predominantly constitute these varieties. Here we aimed to characterize intergenetic recombination and chromosomal imbalances between these A/B/S subgenomes, which often result in copy-number variants (CNVs) leading to changes in gene dosage and phenotype, in a diverse panel of bananas and plantains. This will allow us to characterize varietal lineages better and identify sources of genetic variation. METHODS: We delimited population structure and clonal lineages in a diverse panel of 188 banana and plantain accessions from the most common cultivars using admixture, principal component and phylogenetic analyses. We used new scalable alignment-based methods, Relative Averaged Alignment (RAA) and Relative Coverage, to infer subgenome composition (AA, AAB, etc.) and interspecific recombination. RESULTS: In our panel, we identified ten varietal lineages composed of somatic clones, plus three groups of tetraploid accessions. We identified chromosomal exchanges resulting in gains/losses in chromosomal segments (CNVs), particularly in AAB and ABB varieties. CONCLUSIONS: We demonstrated alignment-based RAA and Relative Coverage can identify subgenome composition and introgressions with similar results to more complex approaches based on single nucleotide polymorphism (SNP) databases. These ab initio species-agnostic methods can be used without sequencing a panel of wild ancestors to find private SNPs, or in recently diverged pools where private SNPs are uncommon. The extensive A/B/S exchanges and the variation in the length of some introgressions between lineages further support multiple foundational events of hybridization and residual backcrossing. Imbalances between A/B/S may have resulted in CNVs and gene dosage variation. Since most edible banana genomes are fixed on time, these CNVs are stable genetic variations probably associated with phenotypic variation for future genetic studies.

Cacao (Theobromacacao L.) climate zones and its associated agrobiodiversity in Arauca, Colombia.

Background: Cacao (Theobromacacao L) is one of the most relevant crops in terms of economy and social rural development in Colombia. Cacao is also an important crop due to its potential to replace illicit crops and it is related to less deforestation and preserves the biodiversity. There are several cacao districts in Colombia, one of these being Arauca. The Department of Arauca is the second largest cocoa producing region in Colombia; however, it is heavily affected by armed conflict. To raise the knowledge and technology available in the region, integrating data on the occurrence of cacao farms with climatic variables becomes a powerful socioeconomic mapping tool for maintaining agrobiodiversity and food security in the region. Consequently, this type of agrodiversity data and agroclimatic approaches help to better manage agrobiodiversity, as in the cacao region of Arauca. These tools are even more relevant in biodiverse regions, such as flooded savannahs and tropical forest ecosystems, which are currently undergoing drastic changes due to agricultural expansion and climate change. One of the knowledge gaps in Colombia´s cacao regions is that there are currently no agroclimatic maps made with a social and scientific approach. This study aimed to provide a database of the spatial distribution of cacao farms in Arauca, as well as agroclimatic maps that identify and locate cacao climate regions in Arauca. We also present a presence-only matrix consisting of twenty-six tree species, or agrobiodiversity, distributed across the study region and specifically associated with the cacao forestry systems in Arauca. New information: We present the first database of both climate and agrobiodiversity data related to cacao farms in Arauca, developed with a research and socioeconomic vision that generated a novel approach for the agroclimatic zoning of cocoa in the Arauca Region and Colombia. Using 1,538 cacao farms at the regional scale, we identified two national and six regional-scale climate and soil regions. The selection at the local scale allowed us to classify 180 cacao farms comprising nine agroclimatic clusters in Arauca. We found twenty-six tree species distributed across the cacao climate zones. This dataset and its related maps also represent the agrobiodiversity of cultivated cacao locally. This is the most complete climate and agrobiodiversity dataset of cacao farms distribution in one of the top cocoa-producing regions in the country. These outputs are crucial because they constitute a baseline for developing research in the biodiversity of agroforestry systems, pests and diseases, pollutant presence, genetics, post-harvest processing and cocoa quality and safety.

Defining a diverse core collection of the Colombian Central Collection of potatoes: a tool to advance research and breeding.

The highly diverse Colombian Central Collection (CCC) of cultivated potatoes is the most important source of genetic variation for breeding and the agricultural development of this staple crop in Colombia. Potato is the primary source of income for more than 100.000 farming families in Colombia. However, biotic and abiotic challenges limit crop production. Furthermore, climate change, food security, and malnutrition constraints call for adaptive crop development to be urgently addressed. The clonal CCC of potatoes contains 1,255 accessions - an extensive collection size that limits its optimal assessment and use. Our study evaluated different collection sizes from the whole clonal collection to define the best core collection that captures the total genetic diversity of this unique collection, to support a characterization more cost-effectively. Initially, we genotyped 1,141 accessions from the clonal collection and 20 breeding lines using 3,586 genome-wide polymorphic markers to study CCC's genetic diversity. The analysis of molecular variance confirmed the CCC's diversity with a significant population structure (Phi=0.359; p-value=0.001). Three main genetic pools were identified within this collection (CCC_Group_A, CCC_Group_B1, and CCC_Group_B2), and the commercial varieties were located across the pools. The ploidy level was the main driver of pool identification, followed by a robust representation of accessions from Phureja and Andigenum cultivar groups based on former taxonomic classifications. We also found divergent heterozygosity values within genetic groups, with greater diversity in genetic groups with tetraploids (CCC_Group_B1: 0.37, and CCC_Group_B2: 0.53) than in diploid accessions (CCC_Group_A: 0.14). We subsequently generated one mini-core collection size of 3 percent (39 entries) and three further core collections sizes of 10, 15, and 20 percent (i.e., 129, 194, and 258 entries, respectively) from the total samples genotyped. As our results indicated that genetic diversity was similar across the sampled core collection sizes compared to the main collection, we selected the smallest core collection size of 10 percent. We expect this 10 percent core collection to be an optimal tool for discovering and evaluating functional diversity in the genebank to advance potato breeding and agricultural-related studies. This study also lays the foundations for continued CCC curation by evaluating duplicity and admixing between accessions, completing the digitalization of data, and ploidy determination using chloroplast count.

Phenotypic and molecular analyses in diploid and tetraploid genotypes of Solanum tuberosum L. reveal promising genotypes and candidate genes associated with phenolic compounds, ascorbic acid contents, and antioxidant activity.

Potato tubers contain biochemical compounds with antioxidant properties that benefit human health. However, the genomic basis of the production of antioxidant compounds in potatoes has largely remained unexplored. Therefore, we report the first genome-wide association study (GWAS) based on 4488 single nucleotide polymorphism (SNP) markers and the phenotypic evaluation of Total Phenols Content (TPC), Ascorbic Acid Content (AAC), and Antioxidant Activity (AA) traits in 404 diverse potato genotypes (84 diploids and 320 tetraploids) conserved at the Colombian germplasm bank that administers AGROSAVIA. The concentration of antioxidant compounds correlated to the skin tuber color and ploidy level. Especially, purple-blackish tetraploid tubers had the highest TPC (2062.41 ± 547.37 mg GAE), while diploid pink-red tubers presented the highest AA (DDPH: 14967.1 ± 4687.79 µmol TE; FRAP: 2208.63 ± 797.35 mg AAE) and AAC (4.52 mg ± 0.68 AA). The index selection allowed us to choose 20 promising genotypes with the highest values for the antioxidant compounds. Genome Association mapping identified 58 SNP-Trait Associations (STAs) with single-locus models and 28 Quantitative Trait Nucleotide (QTNs) with multi-locus models associated with the evaluated traits. Among models, eight STAs/QTNs related to TPC, AAC, and AA were detected in common, flanking seven candidate genes, from which four were pleiotropic. The combination in one population of diploid and tetraploid genotypes enabled the identification of more genetic associations. However, the GWAS analysis implemented independently in populations detected some regions in common between diploids and tetraploids not detected in the mixed population. Candidate genes have molecular functions involved in phenolic compounds, ascorbic acid biosynthesis, and antioxidant responses concerning plant abiotic stress. All candidate genes identified in this study can be used for further expression analysis validation and future implementation in marker-assisted selection pre-breeding platforms targeting fortified materials. Our study further revealed the importance of potato germplasm conserved in national genebanks, such as AGROSAVIA's, as a valuable genetic resource to improve existing potato varieties.

Evolutionary Genetics of Crop-Wild Complexes.

Since Darwin's time, the role of crop wild relatives (CWR), landraces, and cultivated genepools in shaping plant diversity and boosting food resources has been a major question [...].

A new method for the inoculation of Phytophthora palmivora (Butler) into cacao seedlings under greenhouse conditions.

BACKGROUND: The black pod disease affects cacao plantations worldwide; it is caused by the oomycete species of the genus Phytophthora. The resistance of cacao plants to the black pod is commonly evaluated by artificial inoculation of the pathogen and the monitoring of the disease symptoms. However, it is difficult to identify resistant plants because the commonly used methods for the inoculation of the pathogens produce inconsistent results. Therefore, this study aimed to develop an efficient and reliable method to evaluate the resistance of Theobroma cacao seedlings to the infection by Phytophthora palmivora. RESULTS: Seedlings of different cacao genotypes were inoculated with P. palmivora under greenhouse conditions using the previously reported inoculation methods and a newly proposed method, the agar-water solution method. While none of the previously reported methods was effective, the agar-water solution method ensured a 100% seedling infection under greenhouse conditions. The proposed agar-water methodology is fast, simple and reproducible. Furthermore, the evaluation of this method in susceptible (CCN-51) and tolerant (SCA-6) T. cacao genotypes produced the expected contrasting results. CONCLUSIONS: The agar-water solution method presented in this study is an efficient alternative inoculation protocol for the identification of cacao genotypes that are resistant to black pod under greenhouse conditions.

Genome-Wide Association Study Reveals Novel Candidate Genes Associated with Productivity and Disease Resistance to Moniliophthora spp. in Cacao (Theobroma cacao L.).

Cacao (Theobroma cacao L.), the source of chocolate, is one of the most important commodity products worldwide that helps improve the economic livelihood of farmers. Diseases like frosty pod rot caused by Moniliophthora roreri and witches' broom caused by Moniliophthora perniciosa limit the cacao productivity, this can be solved by using resistant varieties. In the current study, we sequenced 229 cacao accessions using genotyping-by-sequencing to examine the genetic diversity and population structure employing 9,003 and 8,131 single nucleotide polymorphisms recovered by mapping against two cacao genomes (Criollo B97-61/B2 v2 and Matina 1-6 v1.1). In the phenotypic evaluation, three promising accessions for productivity and 10 with good tolerance to the frosty pod rot and witches' broom diseases were found. A genome-wide association study was performed on 102 accessions, discovering two genes associated with productivity and seven to disease resistance. The results enriched the knowledge of the genetic regions associated with important cacao traits that can have significant implications for conservation and breeding strategies like marker-assisted selection.

Cacao breeding in Colombia, past, present and future.

Cacao (Theobroma cacao L.) is considered a key crop in Colombian social programs aiming at alleviating rural poverty, promoting peace in post-conflict regions and, replacing crops used for illicit purposes. Colombia is thought to be part of the center of origin of cacao; several germplasm collecting expeditions have been implemented, dating back to the 1940s. Despite that history, the first breeding program based on creating, selecting, and releasing full-sib progenies made extensive use of accessions introduced from other countries as parents. A new breeding strategy was adopted in the 1990s, based on mass selection of promising trees (high-yield and disease-resistant) in farmers' fields, resulting in the selection of clones released to farmers as planting material. In 2012, a new strategy, Recurrent Selection, was adopted by the Colombian Corporation for Agricultural Research, Agrosavia, based on the development of improved populations and allowing the selection of clones at the end of each cycle of recombination. The use of molecular markers is being integrated into this program in order to assist breeders in selecting material. This review provides details about the history and perspectives of the cacao breeding program in Colombia.

Virus-induced gene silencing (VIGS) in cape gooseberry (Physalis peruviana L., Solanaceae) / Silenciamento de genes induzidos por vírus (VIGS) em physalis (Physalis peruviana L., Solanaceae) / Silenciamiento génico inducido por virus (VIGS) en uchuva (Physalis peruviana L., Solanaceae)

Abstract Cape gooseberry (Physalis peruviana, L.) is a herbaceous plant belonging to the Solanaceae family that produces an edible berry appreciated for its nutraceutical and pharmaceutical properties. Its production is often limited by diseases and reproducible fruit quality. Recent studies have reported genes associated with fruit quality and resistance response to the root-infecting fungus Fusarium oxysporum f. sp. physali (Foph,) which causes vascular wilt. In order to standardize a method to validate the biological function of candidate genes in the non-model species P. peruviana, we tested the robust approach in reverse genetics, virus-induced gene silencing (VIGS). In this study, we validated and optimized VIGS using an insert of the phytoene desaturase (PDS) gene in a silencing viral vector generated from tobacco rattle virus (TRV). Leaves infiltrated with Agrobacterium (GV3101 strain) showed photo-bleached segments, which were distinctive for PDS suppression at 7 days post-infection (dpi). More than half of the treated plants showed photo-bleaching, indicating an efficiency rate of 50 % of the VIGS protocol. The results of this study showed that VIGS can be used for future functional gene characterization implicated in the immune response, disease resistance and fruit quality in capegooseberry.

Resumo A physalis (Physalis peruviana, L.) é uma planta herbácea pertencente à família Solanaceae, que produz uma baga comestível apreciada por suas propriedades nutracêuticas e farmacêuticas. Sua produção com frequência se vê limitada devido a enfermidades e baixa reprodutibilidade na qualidade do fruto. Estudos recentes reportaram genes associados com a qualidade do fruto e com a resposta de resistência ao fungo radicular Fusarium oxysporum f. sp. physali (Foph.), que causa esmorecimento vascular. Com a finalidade de padronizar um método para validar a função biológica de genes candidatos na espécie não-modelo P. p ruviana, avaliamos uma aproximação robusta em genética invertida, o sil nciamento de genes induzidos por vírus (VIGS). Neste estudo, validamos e otimizamos o VIGS usando um inserto da fitoeno desaturase (PDS) em um vetor viral de silenciamento produzido a partir do vírus do chocalho do tabaco (TRV). As folhas infiltradas com Agrobacterium (cepa GV3101) mostraram segmentos fotobranqueados, que foram distintivos para a supressão de PDS a 7 dias pós-infecção (dpi). Mais da metade das plantas tratadas mostraram fotobranqueamento, o que indica uma taxa de eficiência de 50 % do procotolo VIGS. Os resultados de este estudo mostraram que o VIGS pode ser usado em caracterizações futuras de genes funcionais implicados na resposta imune, na resistência a enfermidades e na qualidade do fruto de physalis.

Resumen La uchuva (Physalis peruviana, L.) es una planta herbácea perteneciente a la familia de las solanáceas, que produce una baya comestible apreciada por sus propiedades nutracéuticas y farmacéuticas. Su producción con frecuencia se ve limitada debido a enfermedades y a falta de reproducibilidad en la calidad del fruto. Estudios recientes han reportado genes asociados con la calidad del fruto y con la respuesta de resistencia al hongo radicular Fusarium oxysporum f. sp. physali (Foph,), que causa marchitamiento vascular. Con el fin de estandarizar un método para validar la función biológica de genes candidatos en la especie no-modelo P. peruviana, evaluamos la aproximación robusta en genética inversa, el silenciamiento génico inducido por virus (VIGS). En este estudio, validamos y optimizamos el VIGS usando un inserto de la fitoeno desaturasa (PDS) en un vector viral de silenciamiento producido a partir del virus del cascabeleo del tabaco (TRV). Las hojas infiltradas con Agrobacterium (cepa GV3101) mostraron segmentos fotoblanqueados, que fueron distintivos para la supresión de PDS a 7 días pos-infeccion (dpi). Más de la mitad de las plantas tratadas mostraron fotoblanqueo, lo cual indica una tasa de eficiencia del 50 % del protocolo VIGS. Los resultados de este estudio mostraron que el VIGS se puede usar en futuras caracterizaciones de genes funcionales implicados en la respuesta inmune, la resistencia a enfermedad y la calidad del fruto en la uchuva.

Comparative analysis of whole flower transcriptomes in the Zingiberales.

The advancement of next generation sequencing technologies (NGS) has revolutionized our ability to generate large quantities of data at a genomic scale. Despite great challenges, these new sequencing technologies have empowered scientists to explore various relevant biological questions on non-model organisms, even in the absence of a complete sequenced reference genome. Here, we analyzed whole flower transcriptome libraries from exemplar species across the monocot order Zingiberales, using a comparative approach in order to gain insight into the evolution of the molecular mechanisms underlying flower development in the group. We identified 4,153 coding genes shared by all floral transcriptomes analyzed, and 1,748 genes that are only retrieved in the Zingiberales. We also identified 666 genes that are unique to the ginger lineage, and 2,001 that are only found in the banana group, while in the outgroup species Dichorisandra thyrsiflora J.C. Mikan (Commelinaceae) we retrieved 2,686 unique genes. It is possible that some of these genes underlie lineage-specific molecular mechanisms of floral diversification. We further discuss the nature of these lineage-specific datasets, emphasizing conserved and unique molecular processes with special emphasis in the Zingiberales. We also briefly discuss the strengths and shortcomings of de novo assembly for the study of developmental processes across divergent taxa from a particular order. Although this comparison is based exclusively on coding genes, with particular emphasis in transcription factors, we believe that the careful study of other regulatory mechanisms, such as non-coding RNAs, might reveal new levels of complexity, which were not explored in this work.

Colombia a Source of Cacao Genetic Diversity As Revealed by the Population Structure Analysis of Germplasm Bank of Theobroma cacao L.

Beans of the species Theobroma cacao L., also known as cacao, are the raw material to produce chocolate. Colombian cacao has been classified as a fine flavor cacao that represents the 5% of cacao world's production. Colombian genetic resources from this species are conserved in ex situ and in-field germplasm banks, since T. cacao has recalcitrant seeds to desication and long-term storage. Currently, the collection of T. cacao of the Colombian Corporation of Agricultural Research (CORPOICA) has approximately 700 germplasm accessions. We conducted a molecular analysis of Corpoica's cacao collection and a morphological characterization of some accessions with the goal to study its genetic diversity and population structure and, to select interesting accessions for the cacao's breeding program. Phenotypic evaluation was performed based on 18 morphological traits and 4 biochemical traits. PCA analysis of morphological traits explained 60.6% of the total variation in seven components and 100% of the total variation of biochemical traits in four components, grouping the collection in 4 clusters for both variables. We explored 565 accessions from Corpoica's germplasm and 252 accessions from reference populations using 96 single nucleotide polymorphism (SNP) molecular markers. Molecular patterns of cacao Corpoica's collection were obtained amplifying specific alleles in a Fluidigm platform that used integrated circuits of fluids. Corpoica's collection showed highest genetic diversity [Expected Heterozygosity (HE = 0.314), Observed Heterozygosity (HO = 0.353)] that is reduced when reference populations were included in the dataset (HE = 0.294, HO = 0.261). The collection was divided into four clusters based on population structure analysis. Cacao accessions from distinct groups showed some taxonomic concordance and reflected their geographic origins. For instance, accessions classified as Criollo were clearly differentiated in one group and we identified two new Colombian genetic groups. Using a number of allelic variations based on 87 SNP markers and 22 different morphological/biochemical traits, a core collection with a total of 232 accessions was selected as a primary genetic resource for cacao breeders.

Genetic diversity and association mapping in the Colombian Central Collection of Solanum tuberosum L. Andigenum group using SNPs markers.

The potato (Solanum tuberosum L.) is the fourth most important crop food in the world and Colombia has one of the most important collections of potato germplasm in the world (the Colombian Central Collection-CCC). Little is known about its potential as a source of genetic diversity for molecular breeding programs. In this study, we analyzed 809 Andigenum group accessions from the CCC using 5968 SNPs to determine: 1) the genetic diversity and population structure of the Andigenum germplasm and 2) the usefulness of this collection to map qualitative traits across the potato genome. The genetic structure analysis based on principal components, cluster analyses, and Bayesian inference revealed that the CCC can be subdivided into two main groups associated with their ploidy level: Phureja (diploid) and Andigena (tetraploid). The Andigena population was more genetically diverse but less genetically substructured than the Phureja population (three vs. five subpopulations, respectively). The association mapping analysis of qualitative morphological data using 4666 SNPs showed 23 markers significantly associated with nine morphological traits. The present study showed that the CCC is a highly diverse germplasm collection genetically and phenotypically, useful to implement association mapping in order to identify genes related to traits of interest and to assist future potato genetic breeding programs.

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. © 2015 Wiley Periodicals, Inc.

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

BACKGROUND: The 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. RESULTS: Phylogenetic 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. CONCLUSIONS: Our 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.

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.

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.

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).

Multi-locus phylogenies of the genus Barteria (Passifloraceae) portray complex patterns in the evolution of myrmecophytism.

The four species of the central African genus Barteria show variation in habitat and in degree of association with ants. Whereas B. solida, restricted to submontane forests, attracts opportunistic ants to extrafloral nectar, the three other species, found in lowland rainforests (B. fistulosa, B. dewevrei) and in littoral scrub (B. nigritana), possess stem domatia of varying shapes and degrees of specialisation, hosting either non-specific arboreal ants (B. nigritana, some B. dewevrei) or two large species of ants of the genus Tetraponera Smith, 1852 that are specific to some species of Barteria (B. fistulosa, some B. dewevrei). We aimed to investigate whether this variation represents an evolutionary trend toward increasing specialisation of mutualism or the reduction or loss of myrmecophytic traits. For this, we determined phylogenetic relationships within the genus using DNA sequences (primarily nuclear ITS) and microsatellite genotypes (11 loci) on a large sample of individuals, mostly from Cameroon and Gabon. The two types of markers support an initial dichotomy that groups B. dewevrei with B. nigritana and B. fistulosa with B. solida respectively. Within these pairs, species do not appear reciprocally monophyletic. At microsatellite loci, B. nigritana forms a clade embedded within B. dewevrei; and within both B. solida and B. fistulosa, geographical populations show levels of differentiation similar to that observed between populations of B. solida and B. fistulosa. Geographic distance alone does not account for genetic differentiation between species, which indicates reproductive isolation. Divergence in each of the two pairs implies evolutionary transitions in habitat and in myrmecophytism. Specialised mutualism with specific ant species of the genus Tetraponera has been lost in species found in more marginal habitats.