Abstract Cenostigma pyramidale is a native legume of the Brazilian semiarid region which performs symbiotic association with arbuscular mycorrhizal fungi (AMF), being an excellent model for studying genes associated with tolerance against abiotic and biotic stresses. In RT-qPCR approach, the use of reference genes is mandatory to avoid incorrect interpretation of the relative expression. This study evaluated the stability of ten candidate reference genes (CRGs) from C. pyramidale root tissues under salt stress (three collection times) and associated with AMF (three different times of salinity). The de novo transcriptome was obtained via RNA-Seq sequencing. Three algorithms were used to calculate the stability of CRGs under different conditions: (i) global (Salt, Salt+AMF, AMF and Control, and collection times), (ii) only non-inoculated plants, and (iii) AMF (only inoculated plants). HAG2, SAC1, aRP3 were the most stable CRGs for global and AMF assays, whereas HAG2, SAC1, RHS1 were the best for salt stress assay. This CRGs were used to validate the relative expression of two up-regulated transcripts in Salt2h (RAP2-3 and PIN8). Our study provides the first set of reference genes for C. pyramidale under salinity and AMF, supporting future researches on gene expression with this species.

Heat shock (HS) leads to the activation of molecular mechanisms, known as HS-response, that prevent damage and enhance survival under stress. Plants have a flexible and specialized network of Heat Shock Factors (HSFs), which are transcription factors that induce the expression of heat shock proteins. The present work aimed to identify and characterize the Glycine max HSF repertory in the Soybean Genome Project (GENOSOJA platform), comparing them with other legumes (Medicago truncatula and Lotus japonicus) in view of current knowledge of Arabidopsis thaliana. The HSF characterization in leguminous plants led to the identification of 25, 19 and 21 candidate ESTs in soybean, Lotus and Medicago, respectively. A search in the SuperSAGE libraries revealed 68 tags distributed in seven HSF gene types. From the total number of obtained tags, more than 70% were related to root tissues (water deficit stress libraries vs. controls), indicating their role in abiotic stress responses, since the root is the first tissue to sense and respond to abiotic stress. Moreover, as heat stress is related to the pressure of dryness, a higher HSF expression was expected at the water deficit libraries. On the other hand, expressive HSF candidates were obtained from the library inoculated with Asian Soybean Rust, inferring crosstalk among genes associated with abiotic and biotic stresses. Evolutionary relationships among sequences were consistent with different HSF classes and subclasses. Expression profiling indicated that regulation of specific genes is associated with the stage of plant development and also with stimuli from other abiotic stresses pointing to the maintenance of HSF expression at a basal level in soybean, favoring its activation under heat-stress conditions.

Plants experience various environmental stresses, but tolerance to these adverse conditions is a very complex phenomenon. The present research aimed to evaluate a set of genes involved in osmotic response, comparing soybean and medicago with the well-described Arabidopsis thaliana model plant. Based on 103 Arabidopsis proteins from 27 categories of osmotic stress response, comparative analyses against Genosoja and Medicago truncatula databases allowed the identification of 1,088 soybean and 1,210 Medicago sequences. The analysis showed a high number of sequences and high diversity, comprising genes from all categories in both organisms. Genes with unknown function were among the most representative, followed by transcription factors, ion transport proteins, water channel, plant defense, protein degradation, cellular structure, organization & biogenesis and senescence. An analysis of sequences with unknown function allowed the annotation of 174 soybean and 217 Medicago sequences, most of them concerning transcription factors. However, for about 30% of the sequences no function could be attributed using in silico procedures. The establishment of a gene set involved in osmotic stress responses in soybean and barrel medic will help to better understand the survival mechanisms for this type of stress condition in legumes.

Plants have the ability to recognize and respond to a multitude of pathogens, resulting in a massive reprogramming of the plant to activate defense responses including Resistance (R) and Pathogenesis-Related (PR) genes. Abiotic stresses can also activate PR genes and enhance pathogen resistance, representing valuable genes for breeding purposes. The present work offers an overview of soybean R and PR genes present in the GENOSOJA (Brazilian Soybean Genome Consortium) platform, regarding their structure, abundance, evolution and role in the plantpathogen metabolic pathway, as compared with Medicago and Arabidopsis. Searches revealed 3,065 R candidates (756 in Soybean, 1,142 in Medicago and 1,167 in Arabidopsis), and PR candidates matching to 1,261 sequences (310, 585 and 366 for the three species, respectively). The identified transcripts were also evaluated regarding their expression pattern in 65 libraries, showing prevalence in seeds and developing tissues. Upon consulting the SuperSAGE libraries, 1,072 R and 481 PR tags were identified in association with the different libraries. Multiple alignments were generated for Xa21 and PR-2 genes, allowing inferences about their evolution. The results revealed interesting insights regarding the variability and complexity of defense genes in soybean, as compared with Medicago and Arabidopsis.

Worldwide wheat (Triticum aestivum L. em. Thell, 2n = 6X = 42, AABBDD) breeding programs aim to reorganize genotypes to achieve better yields, environmental adaptation and food quality. The necessary interdisciplinarity for breeding purposes requires an accurate choice of the most appropriate cellular and/or molecular strategies available to be integrated with agronomic approaches in order to overcome the genetic limitation of each cultivated species, at each agroecosystem. Cytogenetics has given a great contribution to wheat genetic studies and breeding, due to viability of chromosomal variants because of homoeology among genomes in this allohexaploid species and the genus Triticum. The level of development of cytogenetic techniques achieved over the last 60 years has set wheat apart from other cereal crops in terms of possibilities to introduce genetic material from other species. Cytogenetic approaches have been extensively used in chromosomal mapping and/or resistance gene transference from tribe Triticeae-related species. Monosomic analysis, entire chromosomes engineered through single additions and/or substitutions, reciprocal translocation through radiation or manipulation of homoeologous pairing, as well as synthesis of new amphiploids to allow homologous recombination by chiasmata evolved considerably since the past decades. The association of tissue culture and molecular biology techniques provides bread wheat breeding programs with a powerful set of biotechnological tools. However, knowledge on genetic system components, cytotaxonomical relationships, cytogenetic structure and evolutionary history of wheat species cannot be neglected. This information indicates the appropriate strategy to avoid isolation mechanisms in interspecific or intergeneric crosses, according to the genome constitution of the species the desired gene is to be transferred from. The development of amphiploids as "bridge" species is one of the available procedures to facilitate gene flow between wheat and related species. Since the environment at the center of origin of wheat in Southern Asia is quite different from subtropical environments, Brazilian breeding programs overcome more challenges to adapt wheat crop to biotic and abiotic stresses than some other countries. The germplasm bank of Embrapa Trigo has about 1000 registered entries of Triticum relatives, Aegilops, Secale and Agropyron species supplied from several germplasm banks distributed over the world which were multiplied and/or selected for naturally occurring or artificially inoculated fungal diseases. Since Aegilops squarrosa L. entries showed very good performance, the genetic variability observed in this species was firstly exploited. It is reported here the strategy used for transferring useful genes from Ae. squarrosa (DD, 2n = 14): crossing with tetraploid species (AABB, 2n = 28), rescue and in vitro culture of immature embryos for regeneration of the trihaploid (ABD, 2n = 21) hybrid, and colchicine treatment for genome duplication resulting in the artificial synthesis of hexaploid wheat lines (AABBDD, 2n = 42). Results of 10,739 artificial pollinations involving 28 cross combinations amongst eight T. durum L., T. dicoccum and T. cartlicum tetraploid entries used as female parents and ten selected Ae. squarrosa sources of resistance as male parents are presented here. Immature embryos from 18 cross combinations were recovered and cultured in vitro. Green plantlets from 13 combinations were regenerated. Fertile amphiploids were recovered only from crosses among entries of tetraploid T. durum and diploid Ae. squarrosa. They originated 11 fertile synthetic amphiploid lines from seven different combinations. Useful stem and leaf rust as well as powdery mildew resistance for future use in breeding programs were obtained.