Comparison of ddRADseq and EUChip60K SNP genotyping systems for population genetics and genomic selection in Eucalyptus dunnii (Maiden).

Eucalyptus dunnii is one of the most important Eucalyptus species for short-fiber pulp production in regions where other species of the genus are affected by poor soil and climatic conditions. In this context, E. dunnii holds promise as a resource to address and adapt to the challenges of climate change. Despite its rapid growth and favorable wood properties for solid wood products, the advancement of its improvement remains in its early stages. In this work, we evaluated the performance of two single nucleotide polymorphism, (SNP), genotyping methods for population genetics analysis and Genomic Selection in E. dunnii. Double digest restriction-site associated DNA sequencing (ddRADseq) was compared with the EUChip60K array in 308 individuals from a provenance-progeny trial. The compared SNP set included 8,011 and 19,008 informative SNPs distributed along the 11 chromosomes, respectively. Although the two datasets differed in the percentage of missing data, genome coverage, minor allele frequency and estimated genetic diversity parameters, they revealed a similar genetic structure, showing two subpopulations with little differentiation between them, and low linkage disequilibrium. GS analyses were performed for eleven traits using Genomic Best Linear Unbiased Prediction (GBLUP) and a conventional pedigree-based model (ABLUP). Regardless of the SNP dataset, the predictive ability (PA) of GBLUP was better than that of ABLUP for six traits (Cellulose content, Total and Ethanolic extractives, Total and Klason lignin content and Syringyl and Guaiacyl lignin monomer ratio). When contrasting the SNP datasets used to estimate PAs, the GBLUP-EUChip60K model gave higher and significant PA values for six traits, meanwhile, the values estimated using ddRADseq gave higher values for three other traits. The PAs correlated positively with narrow sense heritabilities, with the highest correlations shown by the ABLUP and GBLUP-EUChip60K. The two genotyping methods, ddRADseq and EUChip60K, are generally comparable for population genetics and genomic prediction, demonstrating the utility of the former when subjected to rigorous SNP filtering. The results of this study provide a basis for future whole-genome studies using ddRADseq in non-model forest species for which SNP arrays have not yet been developed.

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.

On-field phenotypic evaluation of sunflower populations for broad-spectrum resistance to Verticillium leaf mottle and wilt.

Sunflower Verticillium Wilt and Leaf Mottle (SVW), caused by Verticillium dahliae (Kleb.; Vd), is a soil-borne disease affecting sunflower worldwide. A single dominant locus, known as V1, was formerly effective in controlling North-American Vd races, whereas races from Argentina, Europe and an emerging race from USA overcome its resistance. This emphasizes the need for identifying broad-spectrum genetic resistance (BSR) sources. Here we characterize two sunflower mapping populations (MPs) for SVW resistance: a biparental MP and the association MP from the National Institute of Agricultural Technology (INTA), under field growing conditions. Nine field-trials (FTs) were conducted in highly infested fields in the most SVW-affected region of Argentina. Several disease descriptors (DDs), including incidence and severity, were scored across four phenological stages. Generalized linear models were fitted according to the nature of each variable, adjusting mean phenotypes for inbred lines across and within FTs. Comparison of these responses allowed the identification of novel BSR sources. Furthermore, we present the first report of SVW resistance heritability, with estimates ranging from 35 to 45% for DDs related to disease incidence and severity, respectively. This study constitutes the largest SVW resistance characterization reported to date in sunflower, identifying valuable genetic resources for BSR-breeding to cope with a pathogen of increasing importance worldwide.

Midgut Genes Knockdown by Oral dsRNA Administration Produces a Lethal Effect on Cotton Boll Weevil.

The "cotton boll weevil" (Anthonomus grandis Boheman) is a key pest in America whose larval stage develops within the cotton flower bud. During its development, the larva uses the flower bud as food and as a shelter from predators. This behavior limits the effective control through conventional insecticide applications and biocontrol techniques. Increasing genetic information from insects has allowed the development of new control technologies based on the use of RNA interference (RNAi) to design orally delivered double-stranded RNA (dsRNA) strategies. In this study, we evaluated the effect of continuous oral administration of six specific dsRNA in order to identify an effective target gene for RNAi-mediated control of cotton boll weevil. First, six selected A. grandis gene fragments were amplified and cloned to perform in vivo synthesis of the specific dsRNA, and subsequently, larvae and adults were fed with this dsRNA for 2 weeks. Larvae mortality ranged from 40 to 60% depending on the targeted gene sequence. Indeed, α-amylase and cytochrome p450 dsRNAs were the most effective. Oral administration in adults caused smaller but still significant death rates (15-30%). Thus, the results demonstrated RNAi responses depend on life stages and target genes. The dsRNA ingestion was capable of providing knockdown mRNA levels in cotton boll weevil midgut and this effect was significantly higher in the larval stage. In this study, we present a new report of silencing of midgut genes in A. grandis larva induced by continuously feeding with dsRNA. This potential new tool should be further evaluated in cotton boll weevil control strategies.

Transgenic Citrange troyer rootstocks overexpressing antimicrobial potato Snakin-1 show reduced citrus canker disease symptoms.

Citrus canker is a major disease caused by Xanthomonas citri pv. citri. Snakin-1 is an antimicrobial peptide, which was previously shown to be effective against different bacterial and fungal diseases in potato, wheat and lettuce when expressed in transgenic plants. We generated transgenic Citrange Troyer citrus rootstocks constitutively expressing this peptide and 5 different transgenic lines were challenged against virulent X. citri isolates. Challenge assays conducted in vitro using detached leaves and in planta by infiltration revealed a significant reduction of the number and size of canker lesions in some of the transgenic lines.

Exploring sunflower responses to Sclerotinia head rot at early stages of infection using RNA-seq analysis.

Sclerotinia head rot (SHR), caused by the necrotrophic fungus Sclerotinia sclerotiorum, is one of the most devastating sunflower crop diseases. Despite its worldwide occurrence, the genetic determinants of plant resistance are still largely unknown. Here, we investigated the Sclerotinia-sunflower pathosystem by analysing temporal changes in gene expression in one susceptible and two tolerant inbred lines (IL) inoculated with the pathogen under field conditions. Differential expression analysis showed little overlapping among ILs, suggesting genotype-specific control of cell defense responses possibly related to differences in disease resistance strategies. Functional enrichment assessments yielded a similar pattern. However, all three ILs altered the expression of genes involved in the cellular redox state and cell wall remodeling, in agreement with current knowledge about the initiation of plant immune responses. Remarkably, the over-representation of long non-coding RNAs (lncRNA) was another common feature among ILs. Our findings highlight the diversity of transcriptional responses to SHR within sunflower breeding lines and provide evidence of lncRNAs playing a significant role at early stages of defense.

Unveiling the genetic basis of Sclerotinia head rot resistance in sunflower.

BACKGROUND: Sclerotinia sclerotiorum is a necrotrophic fungus that causes Sclerotinia head rot (SHR) in sunflower, with epidemics leading to severe yield losses. In this work, we present an association mapping (AM) approach to investigate the genetic basis of natural resistance to SHR in cultivated sunflower, the fourth most widely grown oilseed crop in the world. RESULTS: Our association mapping population (AMP), which comprises 135 inbred breeding lines (ILs), was genotyped using 27 candidate genes, a panel of 9 Simple Sequence Repeat (SSR) markers previously associated with SHR resistance via bi-parental mapping, and a set of 384 SNPs located in genes with molecular functions related to stress responses. Moreover, given the complexity of the trait, we evaluated four disease descriptors (i.e, disease incidence, disease severity, area under the disease progress curve for disease incidence, and incubation period). As a result, this work constitutes the most exhaustive AM study of disease resistance in sunflower performed to date. Mixed linear models accounting for population structure and kinship relatedness were used for the statistical analysis of phenotype-genotype associations, allowing the identification of 13 markers associated with disease reduction. The number of favourable alleles was negatively correlated to disease incidence, disease severity and area under the disease progress curve for disease incidence, whereas it was positevily correlated to the incubation period. CONCLUSIONS: Four of the markers identified here as associated with SHR resistance (HA1848, HaCOI_1, G33 and G34) validate previous research, while other four novel markers (SNP117, SNP136, SNP44, SNP128) were consistently associated with SHR resistance, emerging as promising candidates for marker-assisted breeding. From the germplasm point of view, the five ILs carrying the largest combination of resistance alleles provide a valuable resource for sunflower breeding programs worldwide.

Potato Snakin-1: an antimicrobial player of the trade-off between host defense and development.

Snakin-1 (SN1) from potato is a cysteine-rich antimicrobial peptide with high evolutionary conservation. It has 63 amino acid residues, 12 of which are cysteines capable of forming six disulfide bonds. SN1 localizes in the plasma membrane, and it is present mainly in tissues associated with active growth and cell division. SN1 is active in vitro against bacteria, fungus, yeasts, and even animal/human pathogens. It was demonstrated that it also confers in vivo protection against commercially relevant pathogens in overexpressing potato, wheat, and lettuce plants. Although researchers have demonstrated SN1 can disrupt the membranes of E. coli, its integral antimicrobial mechanism remains unknown. It is likely that broad-spectrum antimicrobial activity is a combined outcome of membrane disruption and inhibition of intracellular functions. Besides, in potato, partial SN1 silencing affects cell division, leaf metabolism, and cell wall composition, thus revealing additional roles in growth and development. Its silencing also affects reactive oxygen species (ROS) and ROS scavenger levels. This finding indicates its participation in redox balance. Moreover, SN1 alters hormone levels, suggesting its involvement in the complex hormonal crosstalk. Altogether, SN1 has the potential to integrate development and defense signals directly and/or indirectly by modulating protein activity, modifying hormone balance and/or participating in redox regulation. Evidence supports a paramount role to SN1 in the mechanism underlying growth and immunity balance. Furthermore, SN1 may be a promising candidate in preservation, and pharmaceutical or agricultural biotechnology applications.

De novo transcriptome sequencing and SSR markers development for Cedrela balansae C.DC., a native tree species of northwest Argentina.

The endangered Cedrela balansae C.DC. (Meliaceae) is a high-value timber species with great potential for forest plantations that inhabits the tropical forests in Northwestern Argentina.Research on this species is scarce because of the limited genetic and genomic information available. Here, we explored the transcriptome of C. balansae using 454 GS FLX Titanium next-generation sequencing (NGS) technology. Following de novo assembling, we identified 27,111 non-redundant unigenes longer than 200 bp, and considered these transcripts for further downstream analysis. The functional annotation was performed searching the 27,111 unigenes against the NR-Protein and the Interproscan databases. This analysis revealed 26,977 genes with homology in at least one of the Database analyzed. Furthermore, 7,774 unigenes in 142 different active biological pathways in C. balansae were identified with the KEGG database. Moreover, after in silico analyses, we detected 2,663 simple sequence repeats (SSRs) markers. A subset of 70 SSRs related to important "stress tolerance" traits based on functional annotation evidence, were selected for wet PCR-validation in C. balansae and other Cedrela species inhabiting in northwest and northeast of Argentina (C. fissilis, C. saltensis and C. angustifolia). Successful transferability was between 77% and 93% and thanks to this study, 32 polymorphic functional SSRs for all analyzed Cedrela species are now available. The gene catalog and molecular markers obtained here represent a starting point for further research, which will assist genetic breeding programs in the Cedrela genus and will contribute to identifying key populations for its preservation.

Potato snakin-1 gene enhances tolerance to Rhizoctonia solani and Sclerotinia sclerotiorum in transgenic lettuce plants.

Snakin-1 is a cysteine-rich antimicrobial peptide (AMP) isolated from potato tubers, with broad-spectrum activity. It belongs to the Snakin/GASA family, whose members have been studied because of their diverse roles in important plant processes, including defense. To analyze if this defensive function may lead to disease tolerance in lettuce, one of the most worldwide consumed leafy vegetable, we characterized three homozygous transgenic lines overexpressing Snakin-1. They were biologically assessed by the inoculation with the fungal pathogens Rhizoctonia solani and Sclerotinia sclerotiorum both in vitro and in planta at the greenhouse. When in vitro assays were performed with R. solani on Petri dishes containing crude plant extracts it was confirmed that the expressed Snakin-1 protein has antimicrobial activity. Furthermore, transgenic lines showed a better response than wild type in in vivo challenges against R. solani both in chamber and in greenhouse. In addition, two of these lines showed significant in vivo protection against the pathogen S. sclerotiorum in challenge assays on adult plants. Our results show that Snakin-1 is an interesting candidate gene for the selection/breeding of lettuce plants with increased fungal tolerance.

Simplified methodology for large scale isolation of homozygous transgenic lines of lettuce

Background: Lettuce is a globally important leafy vegetable and a model plant for biotechnology due to its adaptability to tissue culture and stable genetic transformation. Lettuce is also crucial for functional genomics research in the Asteraceae which includes species of great agronomical importance. The development of transgenic events implies the production of a large number of shoots that must be differentiated between transgenic and non-transgenic through the activity of the selective agent, being kanamycin the most popular. Results: In this work we adjusted the selection conditions of transgenic seedlings to avoid any escapes, finding that threshold concentration of kanamycin was 75 mg/L. To monitor the selection system, we studied the morphological response of transgenic and non-transgenic seedlings in presence of kanamycin to look for a visual morphological marker. Several traits like shoot length, primary root length, number of leaves, fresh weight, and appearance of the aerial part and development of lateral roots were affected in non-transgenic seedlings after 30 d of culture in selective media. However, only lateral root development showed an early, qualitative and reliable association with nptII presence, as corroborated by PCR detection. Applied in successive transgenic progenies, this method of selection combined with morphological follow-up allowed selecting the homozygous presence of nptII gene in 100% of the analyzed plants from T2 to T5. Conclusions: This protocol allows a simplified scaling-up of the production of multiple homozygous transgenic progeny lines in the early generations avoiding expensive and time-consuming molecular assays.

Integration of transcriptomic and metabolic data reveals hub transcription factors involved in drought stress response in sunflower (Helianthus annuus L.).

KEY MESSAGE: By integration of transcriptional and metabolic profiles we identified pathways and hubs transcription factors regulated during drought conditions in sunflower, useful for applications in molecular and/or biotechnological breeding. Drought is one of the most important environmental stresses that effects crop productivity in many agricultural regions. Sunflower is tolerant to drought conditions but the mechanisms involved in this tolerance remain unclear at the molecular level. The aim of this study was to characterize and integrate transcriptional and metabolic pathways related to drought stress in sunflower plants, by using a system biology approach. Our results showed a delay in plant senescence with an increase in the expression level of photosynthesis related genes as well as higher levels of sugars, osmoprotectant amino acids and ionic nutrients under drought conditions. In addition, we identified transcription factors that were upregulated during drought conditions and that may act as hubs in the transcriptional network. Many of these transcription factors belong to families implicated in the drought response in model species. The integration of transcriptomic and metabolomic data in this study, together with physiological measurements, has improved our understanding of the biological responses during droughts and contributes to elucidate the molecular mechanisms involved under this environmental condition. These findings will provide useful biotechnological tools to improve stress tolerance while maintaining crop yield under restricted water availability.

Phenotyping Sunflower Genetic Resources for Sclerotinia Head Rot Response: Assessing Variability for Disease Resistance Breeding.

Sclerotinia head rot (SHR) is one of the most serious constraints to sunflower (Helianthus annuus L. var. macrocarpus) production worldwide. Here, we evaluated the response to SHR in a sunflower inbred panel from a large INTA germplasm collection, consisting of 137 inbred lines (ILs). Field trials were performed over five consecutive seasons using a twice-replicated randomized complete-block design. Disease incidence, disease severity, incubation period, and area under disease progress curve for disease incidence and severity were determined after controlled inoculation with the pathogen. Statistical analysis using mixed-effect models detected significant differences among ILs for all variables (P < 0.001). In addition, principal component analysis (PCA) and distance-based methods were used to classify the ILs according to their response to SHR, with ILs ALB2/5261 and 5383 emerging as the most resistant. Broad-sense heritability estimates ranged from 20.64% for disease severity to 10.58% for incubation period. The ample phenotypic variability of our collection, along with the moderate heritability estimates, highlight the importance of molecular breeding approaches to gain new insights into the genetic basis of sunflower resistance to SHR. The exhaustive phenotypic characterization presented here provides a reliable set of variables to comprehensively evaluate the disease and identifies two new sources of resistance to SHR.

Temporal expression of CD184(CXCR4) and CD171(L1CAM) identifies distinct early developmental stages of human retinal ganglion cells in embryonic stem cell derived retina.

Human retinal ganglion cells (RGCs) derived from pluripotent stem cells (PSCs) have anticipated value for human disease study, drug screening, and therapeutic applications; however, their full potential remains underdeveloped. To characterize RGCs in human embryonic stem cell (hESC) derived retinal organoids we examined RGC markers and surface antigen expression and made comparisons to human fetal retina. RGCs in both tissues exhibited CD184 and CD171 expression and distinct expression patterns of the RGC markers BRN3 and RBPMS. The retinal progenitor cells (RPCs) of retinal organoids expressed CD184, consistent with its expression in the neuroblastic layer in fetal retina. In retinal organoids CD184 expression was enhanced in RGC competent RPCs and high CD184 expression was retained on post-mitotic RGC precursors; CD171 was detected on maturing RGCs. The differential expression timing of CD184 and CD171 permits identification and enrichment of RGCs from retinal organoids at differing maturation states from committed progenitors to differentiating neurons. These observations will facilitate molecular characterization of PSC-derived RGCs during differentiation, critical knowledge for establishing the veracity of these in vitro produced cells. Furthermore, observations made in the retinal organoid model closely parallel those in human fetal retina further validating use of retinal organoid to model early retinal development.

Sunflower (Helianthus annuus L.).

Sunflower (Helianthus annuus L.) is still considered as a recalcitrant species to in vitro culture and transformation in spite of the publication of different protocols. Here we describe a routine transformation system of this crop which requires mature HA89 genotype seeds and Agrobacterium tumefaciens EHA105 strain for gene delivery, being both easily available. Selection of transformed shoots depends on root development in kanamycin-selective media, instead of shoot color, avoiding selection of escapes. The establishment of this protocol proved successful for the incorporation of both reporter and agronomic important genes and also for the evaluation of the specific expression patterns of different promoters in transgenic sunflower plants. Stable expression of the incorporated transgenes was confirmed by RT-PCR and GUS reporter gene visualization. Stable inheritance of transgenes was successfully followed until T2 generation in several independent lines.

Identification of candidate genes associated with leaf senescence in cultivated sunflower (Helianthus annuus L.).

Cultivated sunflower (Helianthus annuus L.), an important source of edible vegetable oil, shows rapid onset of senescence, which limits production by reducing photosynthetic capacity under specific growing conditions. Carbon for grain filling depends strongly on light interception by green leaf area, which diminishes during grain filling due to leaf senescence. Transcription factors (TFs) regulate the progression of leaf senescence in plants and have been well explored in model systems, but information for many agronomic crops remains limited. Here, we characterize the expression profiles of a set of putative senescence associated genes (SAGs) identified by a candidate gene approach and sunflower microarray expression studies. We examined a time course of sunflower leaves undergoing natural senescence and used quantitative PCR (qPCR) to measure the expression of 11 candidate genes representing the NAC, WRKY, MYB and NF-Y TF families. In addition, we measured physiological parameters such as chlorophyll, total soluble sugars and nitrogen content. The expression of Ha-NAC01, Ha-NAC03, Ha-NAC04, Ha-NAC05 and Ha-MYB01 TFs increased before the remobilization rate increased and therefore, before the appearance of the first physiological symptoms of senescence, whereas Ha-NAC02 expression decreased. In addition, we also examined the trifurcate feed-forward pathway (involving ORE1, miR164, and ethylene insensitive 2) previously reported for Arabidopsis. We measured transcription of Ha-NAC01 (the sunflower homolog of ORE1) and Ha-EIN2, along with the levels of miR164, in two leaves from different stem positions, and identified differences in transcription between basal and upper leaves. Interestingly, Ha-NAC01 and Ha-EIN2 transcription profiles showed an earlier up-regulation in upper leaves of plants close to maturity, compared with basal leaves of plants at pre-anthesis stages. These results suggest that the H. annuus TFs characterized in this work could play important roles as potential triggers of leaf senescence and thus can be considered putative candidate genes for senescence in sunflower.

The P0 protein encoded by cotton leafroll dwarf virus (CLRDV) inhibits local but not systemic RNA silencing.

Plants employ RNA silencing as a natural defense mechanism against viruses. As a counter-defense, viruses encode silencing suppressor proteins (SSPs) that suppress RNA silencing. Most, but not all, the P0 proteins encoded by poleroviruses have been identified as SSP. In this study, we demonstrated that cotton leafroll dwarf virus (CLRDV, genus Polerovirus) P0 protein suppressed local silencing that was induced by sense or inverted repeat transgenes in Agrobacterium co-infiltration assay in Nicotiana benthamiana plants. A CLRDV full-length infectious cDNA clone that is able to infect N. benthamiana through Agrobacterium-mediated inoculation also inhibited local silencing in co-infiltration assays, suggesting that the P0 protein exhibits similar RNA silencing suppression activity when expressed from the full-length viral genome. On the other hand, the P0 protein did not efficiently inhibit the spread of systemic silencing signals. Moreover, Northern blotting indicated that the P0 protein inhibits the generation of secondary but not primary small interfering RNAs. The study of CLRDV P0 suppression activity may contribute to understanding the molecular mechanisms involved in the induction of cotton blue disease by CLRDV infection.

De novo assembly and characterization of leaf transcriptome for the development of functional molecular markers of the extremophile multipurpose tree species Prosopis alba.

BACKGROUND: Prosopis alba (Fabaceae) is an important native tree adapted to arid and semiarid regions of north-western Argentina which is of great value as multipurpose species. Despite its importance, the genomic resources currently available for the entire Prosopis genus are still limited. Here we describe the development of a leaf transcriptome and the identification of new molecular markers that could support functional genetic studies in natural and domesticated populations of this genus. RESULTS: Next generation DNA pyrosequencing technology applied to P. alba transcripts produced a total of 1,103,231 raw reads with an average length of 421 bp. De novo assembling generated a set of 15,814 isotigs and 71,101 non-assembled sequences (singletons) with an average of 991 bp and 288 bp respectively. A total of 39,000 unique singletons were identified after clustering natural and artificial duplicates from pyrosequencing reads.Regarding the non-redundant sequences or unigenes, 22,095 out of 54,814 were successfully annotated with Gene Ontology terms. Moreover, simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 5,992 and 6,236 markers, respectively, throughout the genome. For the validation of the the predicted SSR markers, a subset of 87 SSRs selected through functional annotation evidence was successfully amplified from six DNA samples of seedlings. From this analysis, 11 of these 87 SSRs were identified as polymorphic. Additionally, another set of 123 nuclear polymorphic SSRs were determined in silico, of which 50% have the probability of being effectively polymorphic. CONCLUSIONS: This study generated a successful global analysis of the P. alba leaf transcriptome after bioinformatic and wet laboratory validations of RNA-Seq data.The limited set of molecular markers currently available will be significantly increased with the thousands of new markers that were identified in this study. This information will strongly contribute to genomics resources for P. alba functional analysis and genetics. Finally, it will also potentially contribute to the development of population-based genome studies in the genera.

Characterization of ion currents of murine CD117(pos) stem cells in vitro and their modulation under AT2 R stimulation.

AIM: Hematopoietic stem cells, especially CD117(pos) cells, have been found to possess a regenerative potential in various tissues, in particular cardiac muscle. However, the characterization of the relevant ion currents of stem cells prior to implantation lacks documentation. Activation of angiotensin II type 2 receptor (AT2 R) can lead to further cell differentiation and receptor auto-expression and might thus influence electrophysiological properties of CD117(pos) stem cells. This study was designed to functionally characterize membrane currents of CD117(pos) cells under normal and AT2 R-stimulated conditions. METHODS: CD117(pos) murine bone marrow stem cells were isolated with MACS technique and stimulated for the AT2 R with angiotensin II and losartan for 3-5 days prior to patch-clamp measurements. RT-PCR was used to determine channel expression. Endothelial properties were analysed with immunocytochemistry and acLDL uptake assay. RESULTS: A well-expressed inward rectifying current (IKir ) was identified in cultured CD117(pos) cells. Furthermore, a ZD 7288 (HCN channel blocker)-sensitive current component was isolated. Voltage-dependent potassium currents and chloride currents were less expressed. A small fraction of cells demonstrated voltage- and time-dependent inward currents. In AT2 R-stimulated cells inward rectifying the hyperpolarization-induced inward currents were slightly attenuated on the translational level but showed increased mRNA expression. Cultured CD117(pos) cells express CD31 and VEGFR-2 and significantly increased the uptake of acLDL. CONCLUSIONS: CD117(pos) cells do not have properties of action potential-generating cells and moderately change their excitability during AT2 R stimulation. Electrophysiological and molecular properties of control and AT2 R-stimulated cells point to a differentiation to vascular endothelial cells. This could increase beneficial vascularization in injured tissues.

Microsatellite markers in candidate genes for wood properties and its application in functional diversity assessment in eucalyptus globulus

Background: Functional genetic markers have important implications for genetic analysis by providing direct estimation of functional diversity. Although high throughput sequencing techniques for functional diversity analysis are being developed nowadays, the use of already well established variable markers present in candidate genes is still an interesting alternative for mapping purposes and functional diversity studies. SSR markers are routinely used in most plant and animal breeding programs for many species including Eucalyptus. SSR markers derived from candidate genes (SSR-CG) can be used effectively in co-segregation studies and marker-assisted diversity management. Results: In the present study, eight new non reported SSRs were identified in seven candidate genes for wood properties in Eucalyptus globulus: cinnamoyl CoA reductase (CCR), homocysteine S-methyltransferase (HMT), shikimate kinase (SK), xyloglucan endotransglycosylase 2 (XTH2), cellulose synthase 3 (CesA3), glutathione S-transferase (GST) and the transcription factor LIM1. Microsatellites were located in promoters, introns and exons, being most of them CT dinucleotide repeats. Genetic diversity of these eight CG-derived SSR-markers was explored in 54 unrelated genotypes. Except for XTH2, high levels of polymorphism were detected: 93 alleles (mean of 13.1 sd 1.6 alleles per locus), a mean effective number of alleles (Ne) of 5.4 (sd 1.6), polymorphic information content values (PIC) from 0.617 to 0.855 and probability of Identity (PI) ranging from 0.030 to 0.151. Conclusions: This is the first report on the identification, characterization and diversity analysis of microsatellite markers located inside wood quality candidate genes (CG) from Eucalyptus globulus...