Table of contents: 2018
Plant transformation is a widely used procedure for obtaining transgenic plants and to develop plant models to understand gene function. Plant models such as Nicotiana tabacum are widely used for understanding gene responses to external influences. An important tool in such studies is genetic transformation through infection with Agrobacterium tumefaciens. However, this transformation is often inefficient. Consequently, development and optimization of techniques to promote high rates of seedling regeneration of transgenic tobacco is imperative. The methods tested for infection of tobacco explants consisted of injecting 10 μl of the bacterial culture directly into anodal segment using an insulin syringe (1 mL); bacterial co-cultivation with nodal segments and micro-sectioned leaf disks. Infection through punctures made with a syringe in nodal segments of tobacco and no co-cultivation period was the most efficient in the regeneration process and in obtaining genetically transformed plants, with 88 and 75% success rates, respectively. We obtained an increase of 50% in the transformation rates when compared to previous studies using N. tabacum.
Gold nanoparticles (GNPs) with controlled geometric properties are new options for the management of inflammatory responses and therapy for various diseases, including diabetes mellitus, rheumatoid arthritis, and connective tissue diseases. We examined anti-inflammatory impacts of sugar coated GNPs with different sizes on carrageenan-induced rat paw inflammation. The synthesized GNPs were compared with indomethacin, methotrexate and the commercial gold preparation myochrysine. While methotrexate and myochrysine did not have any significant effects on rat paws, GNPs significantly decreased the inflammatory responses, comparable with indomethacin, a standard anti-inflammatory drug in the rat paw model. There was no harmful effect on metabolic activity of cells treated with GNPs of different sizes. We conclude that sugar coated GNPs are a promising candidate for new options in the management of inflammatory-related diseases.
The adenosine deaminase G22A polymorphism (20q.11.33) affects the level of adenosine deaminase (ADA) expression, which plays an important role in the regulation of intracellular and extracellular concentrations of adenosine. Recent studies reported greater ADA activity in diabetic patients and showed the role of ADA in the modulation of insulin activity and glucose homeostasis. We investigated whether the G22A polymorphism of the ADA gene is associated with type 2 diabetes mellitus (T2DM) in the Palestinian population and assessed the relationship between the G22A variant and fasting plasma glucose (FPG), glycated hemoglobin (HbA1c) and lipid profile among T2DM patients. A total of 231 individuals with T2DM and a control sample of 101 non-diabetic participants were randomly selected from those who were attending United Nations Relief and Works Agency (UNRWA) clinics for treatment and/or follow up. Genomic DNA was extracted from peripheral blood samples and PCR-RFLP was performed to identify the TaqI polymorphism G22A of the ADA gene. No significant differences were observed in the genotype and allele frequencies between T2DM patients and the control group. Yet, among diabetic patients, the GG genotype was significantly associated with higher FPG and HbA1c when compared to the GA+AA genotype but had no influence on blood pressure, BMI or other metabolic parameters. In conclusion, we confirm that the GG genotype of the ADA gene is associated with poor glycemic control in T2DM Palestinians and points to the association of the G22A variant with decreased activity of the ADA enzyme, which is of paramount importance in the pathophysiology of T2DM.
Zungo (ZU), San Pedreño (SP) and Casco de Mula (CM) are Colombian indigenous pig breeds originated from European populations that are well adapted to tropical conditions. These breeds have a great potential to be used in pure and crossbred schemes due to their high reproductive performance and resistance to tropical diseases. We investigated the genetic diversity and genetic structure of these pig breeds using microsatellite molecular markers. Fifty-five ZU, SP and CM animals were genotyped for 10 microsatellites using capillary electrophoresis. The average number of alleles per marker ranged from 5.56±1.88 in ZU to 6.70±1.64 in SP. Observed heterozygosity ranged from 0.68±0.05 in ZU to 0.74±0.03 in SP. Polymorphic information content was high for most of the microsatellites in all breeds. Inbreeding coefficient (FIS) values were low for all microsatellites, with an average of 0.035±0.037. Higher average values closer to and above 0.1 were observed for the fixation index of the global population (FIT) (0.131±0.041) and the coefficient of relatedness between individuals from different populations (FST) (0.099±0.013). Principal components and structure analyses showed a high level of clustering per breed and very low admixture levels between them. The panel of markers used in this study proved to be useful to investigate genetic diversity and pedigree relationships in pig populations. The Colombian pig breeds that we evaluated had a high genetic variability and a well-differentiated genetic structure, which are key for decision making in conservation programs and for the implementation of future animal breeding plans in these populations.
Sine oculis-related homeobox 1 (Six1) and 4 (Six4) transcription factors are expressed in developing and adult muscle. These Six homeoproteins are required to activate the fast-type muscle program in the mouse primary myotome. Whether Six1 and Six4 genes have a role in meat tenderness in Tan sheep has not yet been established. To study the characteristics and mechanisms of meat tenderness in Tan sheep, we sampled the musculus triceps muscle, the longest neck muscles, abdominal muscles, gluteus and biceps femoris muscle and measured Warner-Bratzler shear force and texture profile analysis using a texture analyzer (TMS-Pro), and mRNA expression of Six1 and Six4by real-time PCR, and we performed a correlation analysis. The Warner-Bratzler shear force was significantly different among the five different muscles, but there was no difference in this measure between the triceps and biceps femoris muscles. Hardness, gumminess and chewiness were significantly different among the different muscles. Six1 and Six4 mRNA expression was significantly higher in the longest neck muscles than in the other muscles. There was a significant negative correlation between Six1 mRNA expression and the texture parameters gumminess and chewiness, and a significant positive correlation between Six4 mRNA expression and gumminess and chewiness. Based on these results, we suggest that Six1 and Six4 not only regulate muscle fiber typing, but also meat tenderness in Tan sheep. They could thus be candidate genes for selecting for meat tenderness.
Selection of appropriate genitors in breeding programs increases gains due to the variability found in the divergent groups; this allows quantification of the existing variability, saving time and resources. There are many methods for quantification and evaluation of diversity in population studies, among which we highlight methods that are based on multivariate statistical analyses, such as linear discriminant analysis (LDA) and cluster analysis. Here we propose and evaluate the use of Support Vector machine (SVM) and Artificial Neural Network (ANN) in an attempt to solve the problem of genetic classification of hybrid populations with high degrees of similarity. The results obtained, in terms of the apparent error rate (APER), were compared with those obtained using ANN analysis and LDA. In general, the lowest APER values were associated with scenarios with low degrees of genetic similarity between populations. Specifically, the best results obtained through SVM (ranging from 14.44 to 67.41%) were observed when the exponential radial base kernel function was used. The APERs obtained by the ANN were even lower than those of the linear discriminant function.
Morphological or isozyme markers related to physiological maturation and deteriorative processes are important in the evaluation of seed quality. Two experiments were conducted to examine the possibility of using isozymes as indicators of quality in tobacco seed lots and fruit appearance as an indicator of physiological maturity in tobacco cultivars, based on the physiological and biochemical changes of the seeds. Cultivars CSC 444 and CSC 221 of tobacco fruits were harvested at various maturity stages and their physiological quality was assessed by germination, first count, germination speed index, time to reach 50% germination, cumulative average germination, and seedling emergence. We also assessed the activity of catalase (EC 184.108.40.206 – CAT), esterase (EC 220.127.116.11 – EST), isocitrate dehydrogenase (EC 18.104.22.168 – IDH), malate dehydrogenase (EC 22.214.171.124 – MDH), alcohol dehydrogenase (EC 126.96.36.199 – ADH), endo-β-mannanase (EC 188.8.131.52), and heat-resistant proteins during the process of maturation. Six lots of cultivar CSC 444 were used to differentiate the quality levels between the lots, and their characterization was determined by germination and vigor tests. In addition, we evaluated the enzymatic activity of CAT, EST, ADH, MDH, and heat-resistant proteins. During maturation of the fruits from the partially dark stage, we observed a progressive increase in germination and seed vigor. We concluded that appearance of the fruit is an indicator of fruit maturity and quality in tobacco seeds. The enzymatic profile of ADH matches the physiological potential of the seeds, based on germination and emergence tests. Thus the ADH enzyme indicates the optimum stage to harvest fruits. In the EST and CAT enzymatic pattern analysis, we observed higher activity of these enzymes in lots with lower physiological quality. So the CAT and EST enzymes are biochemical indicators that can assess the deterioration of tobacco seed lots.
We examined the effect of water availability on the nutritional balance of 15 genotypes of the clonal cultivar “Conilon BRS Ouro Preto” of Coffea canephora grown in two contrasting environments in terms of water availability. Biomass production and nutritional balance parameters, based on the deviance from the standard ratio among nutrients for the species, were estimated after 170 days of cultivation in these contrasting environments. The variability of responses among genotypes indicated a favorable for identifying diversity among these genotypes and for selection aiming to explore their nutritional parameters, especially for the concentration of phosphorus and magnesium in green tissues. Cultivation in the environments with low water supply caused losses up to 29% in the biomass production of the young plants (with most severe losses observed for the aerial part); the magnitude of these losses varied among genotypes. Genotypes 125 and 155 accumulated significantly higher amounts of biomass when compared to the others, regardless of the water supply. Overall, the nutritional indexes of the tested genotypes showed greater metabolic inflexibility towards water stress than what is reported for other cultivars of Robusta coffee.
Phosphate (Pi) unavailability is a growth-limiting factor for plants. Under Pi-limited conditions, plants activate molecular mechanisms for better acquisition and utilization of this nutrient. In maize, changes in the expression pattern of several Pi starvation-induced genes, including the A1 coding for dihydroflavonol 4-reductase (DFR) involved in anthocyanin biosynthesis, were identified through microarray analysis. In order to elucidate the molecular determinants with a potential role in P use efficiency, we carried out a study on gene expression analysis of the A1 phosphate responsive gene by northern blot analysis of total RNA from maize genotypes contrasting for Pi efficiency. Two Pi-efficient (L-03 and L-161-1) and five inefficient (L-11, L-16, L-22, L-53, and L-5046) genotypes of maize were grown for 15 days in hydroponic culture in the presence (250 mM Pi) or absence (0 mM Pi) of phosphate. All genotypes showed an increase in anthocyanin accumulation in roots in the absence of Pi (0 mM Pi). The Pi-efficient genotype L-36 and the Pi-inefficient genotypes L-16, L22, and L-5046 showed the highest levels of anthocyanin accumulation. The A1 gene exhibits temporal and spatial expression patterns associated with Pi deficiency. Although there were differences in the expression profile of Pi starvation induced genes, no consistent expression patterns could be associated with either Pi-efficient or Pi-inefficient genotypes. It appears that Pi efficiency in tropical maize is a complex trait mediated by a coordinated action of genes that are either induced or suppressed in response to Pi-deficiency.
Genipa americana (Rubiaceae) is an endemic Amazon-region species that may be subject to inbreeding, since it grows in fragmented environments. To examine this possibility, we examined the genetic diversity and population structure of three G. americana populations naturally grown in Northern Mato Grosso State using SSR markers. Sixty-four individuals were sampled from the three populations: 20 in AFL, 20 in MTP, and 24 in NBD. DNA extraction was performed according to the CTAB method, with modifications. Six SSR primers developed for the species were used. The allele frequency, the observed and expected heterozygosity, polymorphism information content), and the fixation Index were estimated. Molecular variance and principal coordinates analyses were conducted, and the most likely number of groups was inferred using the Structure software to help understand the genetic structure of the populations. The six microsatellite loci used showed 17 alleles, in total, ranging from 2 to 4 alleles per locus, with a mean of 2.83. The expected heterozygosity ranged from 0.35 to 0.67 and remained higher than the observed heterozygosity for all loci. The three populations showed genetic diversity, shared genetic material and presented high inbreeding indexes. Cluster analysis results showed genetic material sharing between populations, as well as a lack of genetic structuring among individuals according to their geographical origins. The molecular characterization revealed that the genetic diversity is higher within than between populations. The three populations had shared genetic material and a high inbreeding index due to low observed heterozygosity. This could be a consequence of the fragmented environment where these populations currently live in, since it reduces the number of G. americana individuals and can increase inbreeding.
We investigated the effects of an allogenic infusion of mesenchymal stem cells derived from adipose tissue for treatment of dogs with neurological sequelae secondary to infection with canine distemper virus. Seven dogs were selected, all of which presented positive PCR for canine distemper and later, after clinical treatment, presented negative PCR but retained at least one post-infection neurological signal. Cell therapy was performed on days 1, 15, and 30; blood samples were taken on days 1 and 30for a hemogram, and the animals were evaluated on days 1, 15, 30, 45, and 180 for walking characteristics, urinary or fecal incontinence, state of consciousness, myoclonus, and occurrence of seizures. A descriptive numerical scale was established for scoring and classifying the various parameters that were evaluated. All the animals demonstrated post-therapy clinical neurological improvement, especially for the parameters walking and urinary and fecal incontinence; this improvement was significant throughout the evaluation period. Allogenic infusion of mesenchymal stem cells derived from adipose tissue in dogs with neurological sequelae secondary to infection by the canine distemper virus proved to be an efficient treatment.
The orchid family Orchidaceae is one of the largest families of angiosperms; it includes approximately 35,000 species, displays a wide range of unique ﬂower shapes and is a valued ornamental crop. Among the various species of orchids, the Phalaenopsis genus is the most widely commercialized among blooming potted plants. Our aim was to provide in-depth sequence information for the various parts of flowers, different time treatments of the protocorm-like bodies and temperature treated shortened stems for flowering pathway regulation, in addition to adding to the available data on normal and peloric mutants. We also obtained transcriptome data from shortened stems under cool temperature for spike induction. The deep sequenced, assembled and annotated information was integrated and built into a database that we named PhalDB. Existing databases, such as OrchidBase, OrchidBase 2.0, Orchidstra, and Orchidstra 2.0 mainly contain information on expressed sequence tags, unigenes, and microRNA only from floral organs of orchid species. These databases do not include information about somaclonal variations and cool temperature treatment, which are important for commercial variety development. PhalDB provides sequence information from 24 samples and covers the above-mentioned tissues or conditions so that comprehensive gene data related to flower development, somaclonal variation and some horticulture traits are available for searching. A user can access DNA level information and miRNA structure, etc. It also provides an opportunity to explore mRNA level information and interactions between genes and miRNA. PhalDB is equipped with a BLAST tool to perform similarity searches among the various gene sequences.
The growing demand for maize creates a challenge for breeders; they need to constantly develop higher yielding and higher quality genotypes. We estimated the most relevant genetic parameters, along with heterosis and variance components. A multivariate approach was used in order to define profiles of narrow sense heritability for yield and nutritional components in half-sibling maize progenies. The applied experimental design was random blocks with a male parent (hybrid tester), five inbred lines (S5) as maternal parents and the progenies (hybrid Top Cross), totaling 11 maize genotypes arranged in six replicates. Agronomic and nutritional characters were evaluated. Half-sibling progenies revealed greater additive genetic contribution to phenotypic expression with grain width and thickness, iron content, total flavonoids and carotenoids, soluble solids, and methionine. Narrow sense heritability values between and within progenies were higher for manganese content, glycine, proline and tryptophan. Regardless of the S5 inbreeding line used, heterosis gains were obtained for ear insertion height, number of grain rows per ear, stalk diameter, zinc content, total carotenoids, soluble solids and pH. Specific heterosis was found for grain yield, glycine, serine, threonine and phenylalanine. The multivariate analysis defined eight profiles of traits according to their genetic tendencies, and indicated narrow sense heritability of the progeny mean as the main reason for this classification.
Heat shock proteins (HSPs) are highly conserved molecules across all plant and animal species. In insects, HSPs are expressed in response to biotic and abiotic stressors and have a well characterized expression pattern in response to heat stress, especially heat shock genes Hsp60, 70 and 90. Temperature affects many aspects of eusocial Hymenoptera, including the stingless bees (Apidae, Meliponini). Consequently, these insects have developed thermal adaptation mechanisms, including thermoregulation. However, this ability decreases when there is deviation from the optimum temperature, compromising colony survival. The study of molecular responses to heat shock stress can be important for the preservation of these pollinizers. We identified and validated in silico the genes encoding HSP60, 70 and 90 in Melipona interrupta, one of the main stingless bees used for honey production in the Amazon region. cDNA fragments of males, workers and queen bees at the white-eyed pupal stage were amplified using degenerate primers. After sequencing, aligning and editing, the sequences were compared with public genomic databases for in silico validation. One fragment of Hsp60, three fragments of Hsp70 and two fragments of Hsp90 were obtained for M. interrupta. These fragments showed 100% similarity with mitochondrial sequences of HSP60 and cytosolic sequences of HSP70 and HSP90 of bees of the genera Apis and Bombus. Therefore, the fragments obtained in this study correspond to parts of HSP60 (mitochondria), HSP70 and HSP90 (cytosol) in white-eyed pupae of M. interrupta. The nucleotide sequences of these fragments did not vary between genders and castes. Our validation in silico of the genes encoding HSPs will be useful for future investigations regarding differential expression of HSPs in response to environmental factors that affect the development, maintenance and survival of stingless bee colonies in the Amazon.
In view of the narrow genetic base of popcorn, probably due to its evolution by selection from flint maize types alone, knowledge about genetic divergence is imperative for the formation of heterotic groups. Thus, our objective was to identify heterotic groups of popcorn lines; we did so by exploiting the representative genotype collection of the Active Popcorn Germplasm Bank of the State University of Northern Rio de Janeiro. Thirty-eight popcorn genotypes from different origins were analyzed by two methodologies to identify divergent groups. In the first method, the genotypic data were processed to determine the number of groups, based on Bayesian clustering algorithms, and two clustering methods (UPGMA and Ward), based on three genetic distance algorithms, weighted index, unweighted index, and an index of genetic distance or dissimilarity, proposed by Smouse and Peakall. The second methodology identified groups based on simultaneous use of morphoagronomic and molecular information and extracting the genetic distance matrix by the Gower algorithm, and later applying UPGMA and Ward clustering methods. The consistency of the clustering methods was evaluated by cophenetic correlation coefficients. The significance of these coefficients was examined by the Mantel test. There was significant genetic variability among corn popcorn accesses at morphological and molecular levels. There also was agreement between multivariate clustering techniques, mainly when using genotypic data provided by microsatellite markers. heterotic groups were identified; these were formed mainly according to the origin of each genotype and/or geographic origin. We found that there is sufficient heterosis to develop new cultivars.