Optimized method for differential gene expression analysis in non-model species: Case of Cedrela odorata L.

The following protocol introduces a targeted methodological approach of differential gene expression analysis, which is particularly beneficial in the context of non-model species. While we acknowledge that biological complexity often involves the interplay of multiple genes in any given biological response our method provides a strategy to streamline this complexity, enabling researchers to focus on a more manageable subset of genes of interest. In this context, red cedar transcriptome (Cedrela odorata L.) and known or hypothetical genes related to the response to herbivory were used as reference. The protocol key points are:•Implementation of a transcriptome thinning process to eliminate redundant and non-coding sequences, optimizing the analysis and reducing processing time.•Use of a custom gene database to identify and retain coding sequences with high precision.•Focus on specific genes of interest, allowing a more targeted analysis for specific experimental conditions. This approach holds particular value for pilot studies, research with limited resources, or when rapid identification and validation of candidate genes are needed in species without a reference genome.

The Best of Both Worlds-Streptomyces coelicolor and Streptomyces venezuelae as Model Species for Studying Antibiotic Production and Bacterial Multicellular Development.

Streptomyces bacteria have been studied for more than 80 years thanks to their ability to produce an incredible array of antibiotics and other specialized metabolites and their unusual fungal-like development. Their antibiotic production capabilities have ensured continual interest from both academic and industrial sectors, while their developmental life cycle has provided investigators with unique opportunities to address fundamental questions relating to bacterial multicellular growth. Much of our understanding of the biology and metabolism of these fascinating bacteria, and many of the tools we use to manipulate these organisms, have stemmed from investigations using the model species Streptomyces coelicolor and Streptomyces venezuelae. Here, we explore the pioneering work in S. coelicolor that established foundational genetic principles relating to specialized metabolism and development, alongside the genomic and cell biology developments that led to the emergence of S. venezuelae as a new model system. We highlight key discoveries that have stemmed from studies of these two systems and discuss opportunities for future investigations that leverage the power and understanding provided by S. coelicolor and S. venezuelae.

Peculiarities of the Transformation of Asteraceae Family Species: The Cases of Sunflower and Lettuce.

The Asteraceae family is the largest and most diversified family of the Angiosperms, characterized by the presence of numerous clustered inflorescences, which have the appearance of a single compound flower. It is estimated that this family represents around 10% of all flowered species, with a great biodiversity, covering all environments on the planet, except Antarctica. Also, it includes economically important crops, such as lettuce, sunflower, and chrysanthemum; wild flowers; herbs, and several species that produce molecules with pharmacological properties. Nevertheless, the biotechnological improvement of this family is limited to a few species and their genetic transformation was achieved later than in other plant families. Lettuce (Lactuca sativa L.) is a model species in molecular biology and plant biotechnology that has easily adapted to tissue culture, with efficient shoot regeneration from different tissues, organs, cells, and protoplasts. Due to this plasticity, it was possible to obtain transgenic plants tolerant to biotic or abiotic stresses as well as for the production of commercially interesting molecules (molecular farming). These advances, together with the complete sequencing of lettuce genome allowed the rapid adoption of gene editing using the CRISPR system. On the other hand, sunflower (Helianthus annuus L.) is a species that for years was considered recalcitrant to in vitro culture. Although this difficulty was overcome and some publications were made on sunflower genetic transformation, until now there is no transgenic variety commercialized or authorized for cultivation. In this article, we review similarities (such as avoiding the utilization of the CaMV35S promoter in transformation vectors) and differences (such as transformation efficiency) in the state of the art of genetic transformation techniques performed in these two species.

Transcriptome Analysis of Melocactus glaucescens (Cactaceae) Reveals Metabolic Changes During in vitro Shoot Organogenesis Induction.

Melocactus glaucescens is an endangered cactus highly valued for its ornamental properties. In vitro shoot production of this species provides a sustainable alternative to overharvesting from the wild; however, its propagation could be improved if the genetic regulation underlying its developmental processes were known. The present study generated de novo transcriptome data, describing in vitro shoot organogenesis induction in M. glaucescens. Total RNA was extracted from explants before (control) and after shoot organogenesis induction (treated). A total of 14,478 unigenes (average length, 520 bases) were obtained using Illumina HiSeq 3000 (Illumina Inc., San Diego, CA, USA) sequencing and transcriptome assembly. Filtering for differential expression yielded 2,058 unigenes. Pairwise comparison of treated vs. control genes revealed that 1,241 (60.3%) unigenes exhibited no significant change, 226 (11%) were downregulated, and 591 (28.7%) were upregulated. Based on database analysis, more transcription factor families and unigenes appeared to be upregulated in the treated samples than in controls. Expression of WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) and CALMODULIN (CaM) genes, both of which were upregulated in treated samples, was further validated by real-time quantitative PCR (RT-qPCR). Differences in gene expression patterns between control and treated samples indicate substantial changes in the primary and secondary metabolism of M. glaucescens after the induction of shoot organogenesis. These results help to clarify the molecular genetics and functional genomic aspects underlying propagation in the Cactaceae family.

The Relevance of a Physiological-Stage Approach Study of the Molecular and Environmental Factors Regulating Seed Germination in Wild Plants.

Germination represents the culmination of the seed developmental program and is affected by the conditions prevailing during seed maturation in the mother plant. During maturation, the dormancy condition and tolerance to dehydration are established. These characteristics are modulated by the environment to which they are subjected, having an important impact on wild species. In this work, a review was made of the molecular bases of the maturation, the processes of dormancy imposition and loss, as well as the germination process in different wild species with different life histories, and from diverse habitats. It is also specified which of these species present a certain type of management. The impact that the domestication process has had on certain characteristics of the seed is discussed, as well as the importance of determining physiological stages based on morphological characteristics, to face the complexities of the study of these species and preserve their genetic diversity and physiological responses.

How can an infaunal brooding echinoid be maintained in the laboratory? A case study with Cassidulus mitis (Echinoidea: Cassiduloida) / ¿Cómo se puede mantener un equinoide infantil en el laboratorio? Un estudio de caso con Cassidulus mitis (Echinoidea: Cassiduloida)

Introduction: Cassiduloids play a prominent role in echinoid evolutionary history because they probably are the ancestral group of clypeasteroids. Some extant species are brooding and rare in the environment. Consequently, there are no studies on their maintenance in the laboratory. Objective: Establish an efficient aquarium system for C. mitis, endemic to Brazil, for ontogenetic studies. Methods: Four aquarium systems were built, with 3 replicates each one: (1) with seawater flow [F]; (2) with seawater flow and air injection into sediment [FA]; (3) without seawater flow but with air injection into the sediment [A]; and (4) without both seawater flow and air injection into the sediment [C]. Each experimental aquarium (three per treatment) had two adults. Each of the two sets of experiments lasted about 60 days. Results: We observed low mortality in the first 30 days in all systems and, after 30 days, it was higher in those with air-pumped into the sediment (system A in the first set of experiments, and system FA in the second one). Conclusions: For experiments lasting 30 days, our four systems are suitable. For longer periods, we recommend aquaria with seawater flow and without air-pumps into the sediment.

Introducción: Los casiduloides desempeñan un papel destacado en la historia evolutiva de los equinoides porque probablemente son el grupo ancestral de clipeasteroides. Algunas especies existentes son inquietantes y raras en el medio ambiente. En consecuencia, no existen estudios sobre su mantenimiento en laboratorio. Objetivo: Establecer un sistema de acuario eficiente para C. mitis, endémica de Brasil, para estudios ontogenéticos. Métodos: Se construyeron cuatro sistemas de acuarios, con 3 réplicas cada uno: (1) con flujo de agua de mar [F]; (2) con flujo de agua de mar e inyección de aire en el sedimento [FA]; (3) sin flujo de agua de mar pero con inyección de aire en el sedimento [A]; y (4) sin flujo de agua de mar ni inyección de aire en el sedimento [C]. Cada acuario experimental (tres por tratamiento) tenía dos adultos. Cada uno de los dos conjuntos de experimentos duró aproximadamente 60 días. Resultados: Observamos una baja mortalidad en los primeros 30 días en todos los sistemas y, después de 30 días, fue mayor en aquellos con aire bombeado al sedimento (sistema A en el primer conjunto de experimentos y sistema FA en el segundo). Conclusiones: Para experimentos de 30 días, nuestros cuatro sistemas son adecuados. Para períodos más largos, recomendamos acuarios con flujo de agua de mar y sin bombas de aire en el sedimento.

Eutrema salsugineum (Cruciferae) new to Mexico: a surprising generic record for the flora of Middle America.

The paper reports Eutrema salsugineum as a novelty to the flora of Mexico and Middle America in general. The finding stands ca. 1600 km apart from the closest known locality in the Rocky Mountains of Colorado, USA. The species is considered native to NW Mexico and its late discovery in the region is presumably explained by its tiny habit, early flowering time, and subephemeral life cycle. The phylogenetic position of this Mexican population in a haplotype network based on the chloroplast DNA fragment psbA-trnH confirms this hypothesis and also suggests, in contrast to the previously held viewpoint, multiple colonizations of North American continent from Asia.

Anonymous nuclear markers data supporting species tree phylogeny and divergence time estimates in a cactus species complex in South America.

Supportive data related to the article "Anonymous nuclear markers reveal taxonomic incongruence and long-term disjunction in a cactus species complex with continental-island distribution in South America" (Perez et al., 2016) [1]. Here, we present pyrosequencing results, primer sequences, a cpDNA phylogeny, and a species tree phylogeny.

Perspectives for a Framework to Understand Aril Initiation and Development.

A differentiated structure called "aril" has been described in seeds of several plant species during the course of evolution and might be considered as a supernumerary integument. Besides its ecological function in seed dispersal, the structure also represents a relevant character for systematic classification and exhibits important properties that impart agronomic value in certain species. Little is known about the molecular pathways underlying this morphological innovation because it is absent in currently used model species. A remarkable feature of the seeds of Passiflora species is the presence of a conspicuous aril. This genus is known for the ornamental, medicinal, and food values of its species. In view of the molecular resources and tools available for some Passiflora species, we highlight the potential of these species as models for developmental studies of the aril.

Anonymous nuclear markers reveal taxonomic incongruence and long-term disjunction in a cactus species complex with continental-island distribution in South America.

The Pilosocereus aurisetus complex consists of eight cactus species with a fragmented distribution associated to xeric enclaves within the Cerrado biome in eastern South America. The phylogeny of these species is incompletely resolved, and this instability complicates evolutionary analyses. Previous analyses based on both plastid and microsatellite markers suggested that this complex contained species with inherent phylogeographic structure, which was attributed to recent diversification and recurring range shifts. However, limitations of the molecular markers used in these analyses prevented some questions from being properly addressed. In order to better understand the relationship among these species and make a preliminary assessment of the genetic structure within them, we developed anonymous nuclear loci from pyrosequencing data of 40 individuals from four species in the P. aurisetus complex. The data obtained from these loci were used to identify genetic clusters within species, and to investigate the phylogenetic relationship among these inferred clusters using a species tree methodology. Coupled with a palaeodistributional modelling, our results reveal a deep phylogenetic and climatic disjunction between two geographic lineages. Our results highlight the importance of sampling more regions from the genome to gain better insights on the evolution of species with an intricate evolutionary history. The methodology used here provides a feasible approach to develop numerous genealogical molecular markers throughout the genome for non-model species. These data provide a more robust hypothesis for the relationship among the lineages of the P. aurisetus complex.