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Table of contents: 2020

Research Article

Fetal sexing from maternal plasma is aimed at early determination of fetal sex, and it is possible to detect in the maternal plasma specific alleles of the fetus, which are inherited paternally. In this case, specific Y chromosome sequences are amplified by PCR. This PCR technique is a useful tool for determining fetal sex, with no risk to the fetus. We determined fetal sex by molecular sexing technique at different gestational stages and compared the results with obstetric ultrasound information, in pregnant women between the 8th and 13th weeks of gestation, who were over 18 years of age. Among the 32 samples collected, 21 were females and 11 were males. Four samples showed false negative results, i.e., the Y chromosome DNA was not detected; however the fetus was male. All results incompatible with true fetal sex were collected from pregnant women at the 8th gestational week. In all four samples, the PCR failed to detect the Y chromosome. The determination of fetal sex from the 9th gestational week on had a satisfactory sensitivity index. The sensitivity and specificity of the tests increase with gestational age, making the test safe and with low probability of false negatives. The maternal cell-free plasma noninvasive test proved accurate for sex detection; it can be considered a practical alternative to traditional invasive diagnostic procedures.

Genet. Mol. Res. 19(2): GMR18559
DOI: 10.4238/gmr18559
Research Article

Phenotypic and genotypic correlations of agronomic traits with resistance to phytopathogens can help identify indirect selection criteria for corn yield. The Brazilian corn cultivar NAP5 is resistant to leaf spot and blight disease, while NAP7 is resistant to rust disease. Trials were conducted in two states from February to July 2015. The following traits were evaluated: GY – grain yield, PH - plant height, EH - ear height, MF – days to male flowering, NP - number of plants, NE - number of ears, BP - number of broken plants, LP - number of lodged plants, KW – kernel weight, HT - resistance to the fungus Exserohilum turcicum, WS (white spot) - resistance to Pantoea ananatis, CS - resistance to corn stunt, PP - resistance to Puccinia polysora and CZ - resistance to Cercospora zeae-​​maydis. The NAP5 population showed high genetic correlations between GY and PH, NP, NE and CS in Jataí, GO. In Uberlândia, MG, NAP5 presented high correlations between GY and PH, NP, NE, WS and CS. For the NAP7 population, genetic correlations were high between GY and CS in Uberlândia and between GY and NE in Jataí. Genetic gains for GY by direct selection ranged from 29 to 31%. The influence of the location was low. NE had the greatest effect on grain yield. For the NAP5 population, indirect selection for yield through selection for NE gave satisfactory genetic gain. The variable NE affected KW, and could be used for indirect selection, though direct selection provides greater gains. The heritability estimate for GY (0.77) in the NAP5 population was higher than for NE (0.61). With direct selection, it is possible to obtain gains of up to 31% in GY. High genetic correlations were also observed in the NAP5 population between NE, GY and CS.

Genet. Mol. Res. 19(2): GMR18408
DOI: 10.4238/gmr18408
Research Article

Bees are important pollinating agents and are an integral part of food production and plant maintenance. It is useful to investigate mitochondrial polymorphisms in bees to obtain information that may be relevant to conservation strategies. We examined mitochondrial haplotypes and evaluated genetic diversity and differentiation in the native stingless bees Tetragonisca angustula and Tetragonisca weyrauchi using PCR-restriction fragment length polymorphism. Worker bees were collected from nests in Rondônia state. After isolating DNA, analyses were performed using 10 pairs of heterologous primers for T. weyrauchi, amplifying various regions of mitochondrial DNA (mtDNA). Primers and restriction enzymes were used for the first time for T. weyrauchi but had already been used in other studies with T. angustula. Only four of the tested primers (primer 1 - ND2 and COI; primer 2 - COI; primer 8 - 16S and 12S, and primer 9 - COII) were used for further analyses. For restriction analysis of the amplified regions, 13 enzymes were tested. Primer 1 - (ND2, COI) allowed the identification of a fragment approximately 2,400 bp in size in T. angustula and T. weyrauchi. Fragment cleavage was accomplished using EcoRI and EcoRV enzymes. An approximately 1,850 bp fragment was amplified in the two species using region (COI) primer 2. Using the EcoRV enzyme, cleavage was confirmed only in T. angustula individuals; however, it was observed in both species using the HinfI enzyme. Amplification of the region, using 16S and 12S - primer 8, generated two fragments (1,850 and 350 bp in size), and cleavage was observed in both species using EcoRV, RsaI, and PstI enzymes; however, the XbaI enzyme cleaved in T. weyrauchi alone. Amplification of DNA, using (COII) primer 9, generated a 1,000 bp fragment. The cleavage was performed using ClaI and HinfI enzymes in T. angustula. Bayesian inference analysis showed that mtDNA of T. angustula has a greater genetic variability than that of T. weyrauchi. The variations observed by the analysis of mtDNA using PCR-RFLP showed differences in mtDNA in the two species.

Genet. Mol. Res. 19(2): GMR18495
DOI: 10.4238/gmr18495
Research Article

Atherosclerosis is a multifactorial chronic-inflammatory disease related to endothelial aggression to the intima layer of medium and large caliber arteries. Hyperlipidemia and atherosclerosis cause eNOS to lose its function, producing superoxide and leading to endothelial dysfunction. The nitric oxide derived from eNOS is anti-atherogenic. Single nucleotide polymorphisms in the promoter region reduce its activity and predispose individuals to cardiovascular disease. We analyzed the T786C polymorphism of eNOS in atherosclerotic patients under statin treatment, to determine the clinical importance of this type of genetic variation. The study of this polymorphism in atherosclerotic patients with stents could help predict the probability of restenosis.  We collected 79 peripheral blood samples from patients diagnosed with atherosclerosis undergoing statin treatment. These included 35 stent patients and 44 patients without stents. The TC genotype was prevalent in stent patients who smoke but there was no significant relation between the T786C polymorphism and restenosis. Based on an in silico approach through molecular modeling and molecular docking, we found that statins stabilize the eNOS protein. Seven amino acid residues in the eNOS binding pocket interact with the statin molecule; this family of drugs acts by stabilizing the eNOS protein. Thus, the use of such drugs may help reduce the risk of restenosis.

Genet. Mol. Res. 19(2): GMR18539
DOI: 10.4238/gmr18539
Research Article

Ginger (Zingiber officinale Roscoe) is a self-incompatible plant and has high rates of infertility; its genetic diversity only occurs via processes of mutation and natural selection. Since ginger is important as a condiment and as an herbal medicine, understanding its diversity to a greater degree can contribute both to its conservation and to its use in breeding programs. Considering the importance of the species and the characterization of material from on-farm conservation, this study aimed to evaluate, by means of ISSRs (Inter Simple Sequence Repeats) molecular markers of genetic diversity among individuals obtained in 19 urban and rural backyards in Alta Floresta, Mato Grosso state, Brazil. For the extraction of total DNA, the CTAB method was used and the amplifications were performed using nine ISSR primers. The UPGMA clustering method, when compared with the Ward and Nearest Neighbor methods, best showed genetic diversity, and the Bayesian analysis defined two distinct groups in the Z. officinale germplasm that was evaluated. The ISSR primers amplified a total of 78 fragments and revealed 88% polymorphism. The PIC varied between 0.40 and 0.87, with an average of 0.70, and was classified as median in the detection of polymorphism. The most dissimilar individuals were AF06 and AF12, while the least dissimilar were AF06 and AF04. The Nei (He) and Shannon (I) diversity indices demonstrated genetic variability within the two groups formed from the Bayesian analysis, and AMOVA indicated that the genetic diversity was greater within the groups (53.06%) than among them (46.94%). The absence of duplicates among the individuals analyzed indicates that they can be used to form an active germplasm collection with the aim of conserving and maintaining the genetic diversity of the species.

Genet. Mol. Res. 19(2): GMR18576
DOI: 10.4238/gmr18576
Research Article

The soybean stem fly (Melanagromyza sojae) is a widely distributed and highly damaging soybean pest, recently introduced to Brazil and neighboring countries. The bioecology of this pest under South American growing conditions is largely unknown, including how infesting populations survive throughout the winter. Fly larvae collected in August 2019 from Persian clover Trifolium resupinatum in Santa Maria, southern Brazil, were identified as M. sojae via molecular characterization. Persian clover is commonly grown as a cattle forage crop in this region. This is the first report of M. sojae occurring on T. resupinatum, and of any overwintering host available to the pest in the New World. This finding will help understand the bioecology of M. sojae populations in Brazil, so that adequate pest management strategies can be planned for this invasive pest.

Genet. Mol. Res. 19(2): GMR18627
DOI: 10.4238/gmr185627
Research Article

The Asteraceae have high ecological importance in the threatened Atlantic Forest biome, as most species are pioneers and nectar-producing plants. Using population genetic data could help develop adequate conservation strategies for species and biomes. However, no genetic data or microsatellite markers are available for most Atlantic Forest native Asteraceae species. In their absence, heterologous microsatellite markers could help conduct population genetic studies of less studied species. We evaluated the transferability and utility for population studies of 15 anonymous microsatellite primers pairs developed from other Asteraceae to four others, three of which are important in folk medicine (Baccharis milleflora, Baccharis articulata, Baccharis dracunculifolia) and a toxic species of veterinary importance (Senecio brasiliensis). We found that the microsatellite primers had high transferability to phylogenetically close Asteraceae species. Transferability rates were below those reported for other plant families. The transferred microsatellite primers gave low polymorphism frequencies and high null allele frequencies in the populations. A major factor contributing to this low transferability and high frequency of null alleles is probably the high genetic variation of tropical Asteraceae.

Genet. Mol. Res. 19(2): GMR18546
DOI: 10.4238/gmr18546
Research Article

The study of microorganisms that inhabit the interior of plants (endophytes) has acquired great importance because of their potential to produce bioactive metabolites. Acmella ciliata (known in Brazil as jambu) is a native herb of South America, used in regional gastronomy and folk medicine in Amazonas - Brazil, has antibacterial properties, and may be a useful host for bioactive endophytes. We isolated endophytic microorganisms from A. ciliata and evaluated their ability to inhibit pathogenic fungi. We isolated 56 fungi and 39 endophytic bacteria, most of them from the leaves. The endophyte isolates were then tested in antagonism assays against the phytopathogens Fusarium decemcellulare and Colletotrichum gloeosporioides. The best antagonism index values against C. gloeosporioides were obtained with the endophytic fungus UEA-253 (40%), and the endophytic bacterium UEA-135 (46.3%). The highest antagonism index values were obtained against F. decemcellulare with the fungus UEA-234 (47.2%), and the bacterium UEA-135 (44.8%). The endophytic fungi with inhibitory bioactivity belong to the genera Curvularia, Colletotrichum, Plectosphaerella and Sordariomycetes, while the endophytic bacteria belong to the genera Bacillus, Pseudomonas and Enterobacter. We conclude that the endophytic fungi and bacteria isolated from A. ciliata have potential for use in the biocontrol of F. decemcellulare and C. gloeosporioides.

Genet. Mol. Res. 19(2): GMR18570
DOI: 10.4238/gmr18570
Research Article

Cross-pollination and gametophytic self-incompatibility reduce the stability of Coffea canephora genotypes. This is an important crop for Brazil, the largest producer of this type of coffee and also a major exporter. The study of biometric characteristics is essential to assist in the selection of promising plant materials. We examined the diversity of morpho-agronomic traits of genotypes of C. canephora cv. Conilon through the evaluation of branch and leaf parameters. Assessments included plagiotropic branch length, number of nodes in plagiotropic branches, distance between nodes in plagiotropic branches, orthotropic branch length, number of nodes in orthotropic branch, distance between nodes in orthotropic branch, plant height, canopy diameter, leaf length, leaf width, and leaf area in two periods. The data from the 43 coffee genotypes were tested by multivariate and cluster analyses. Six groups were formed by the Tocher optimization method, and five groups by the unweighted pair group method with arithmetic mean (UPGMA) hierarchical method, suggesting an important genetic variability among plant materials. Both Tocher optimization and UPGMA hierarchical methods were consistent for clustering the genotypes, ordering them in six and five dissimilar groups, respectively, with genotypes 25 and 37 standing out with the greatest dissimilarity, constituting isolated groups by both methods. Pearson’s correlation ranged from very weak to very strong, positive and negative, among the characteristics, as also shown by principal component analyses. These analyses indicated the morpho-agronomic traits with a greater degree of correlation, assisting in the choice of promising plant materials. The genetic parameters estimates demonstrate genetic variability and thus breeding potential within the Conilon coffee genotypes studied. These results emphasize the usefulness of biometric evaluations as a tool for the identification and breeding of genotypes to compose new Conilon coffee cultivars.

Genet. Mol. Res. 19(2): GMR18541
DOI: 10.4238/gmr18541
Research Article

Due to the increasing popularity of consuming only plant products, including vegetarianism and more recently veganism, vegetables and fruits have gradually become an increasingly important component of the human diet, worldwide. The large diversity of Brazilian tropical fruits, such as murici, Byrsonima crassifolia, (Malpighiaceae), associated with a dearth of information concerning this and other exotic species, has stimulated research on techniques to obtain increases in productivity, inserting these fruit species into regional and national agribusiness. We evaluated the variability of B. crassifolia genotypes and quantified the relative contribution of 12 morphological traits of its fruits and seeds. Ripe fruits were collected from 20 different genotypes (trees. The fruits were collected during the harvest season from December 2017 to January 2018. Twenty fruits of each genotype were selected; that were visually appealing, whole and without deformation, totaling 400 samples. We measured the length, width, thickness, weight, and volume of the fruits, the length, width, thickness and weight of the seed and the thickness and weight of the pulp. The data were obtained with a digital caliper and the degrees brix measured with a refractometer. Multivariate analyses were made with UPGMA hierarchical methods and Tocher optimization, using the standardized average Euclidean distance as a measure of dissimilarity, and principal components analysis (PCA). Four distinct groups were formed based on the data. The first three principal components explained 87.51% of the total variation. The brix measurement was the trait with the greatest contribution to the differentiation of the genotypes.

Genet. Mol. Res. 19(2): GMR18620
DOI: 10.4238/gmr18620
Research Article

Even though dengue remains a major threat in Malaysia, information on insecticide resistance of vectors, along with the underlying genetic basis of such resistance, and the impact of a natural flooding disaster remains sparse. Kelantan was one of the states of Malaysia severely affected by monsoon flooding in December 2014. We examined the resistance profile of Aedes mosquitoes in after the big flood, comparing the susceptibility of Ae. aegypti and Ae. albopictus from flooded and unflooded areas. We also sought to validate a simple molecular assay for detecting knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) gene of the mosquitoes. Mosquito immatures were collected by using ovitraps in Kampung Baru, Pasir Pekan, Tumpat (flooded area), and Bandar Baru Kubang Kerian, Kubang Kerian (unflooded area) five months after the flood disaster. The samples were reared to adult mosquitoes and bio-assayed following World Health Organization (WHO) protocol against deltamethrin (0.05%) and pirimiphos-methyl (0.25%) to evaluate their susceptibility. The DNA molecular assays focused on amino acid substitution in domain II (S989, I1011, L1014, and V1016) and domain III (F1534C) in segment 6 of the VGSC gene. Aedes aegypti and Ae. albopictus from both locations were found to be susceptible to pirimiphos-methyl (mortality >98%). A high level of resistance to deltamethrin was detected in Ae. Aegypti; the unflooded area mosquitoes had significantly lower mortality (17%) than the flooded area (74%). Investigation of kdr mutation showed F1534C substitution in the VGSC gene of Ae. aegypti from the flooded and unflooded areas, with an insignificant difference in frequency of 83 and 75% (P > 0.05). This mutation was not detected in Ae. albopictus.

Genet. Mol. Res. 19(2): GMR18604
DOI: 10.4238/gmr18604
Research Article

Psidium guajava (guava) is a fruit tree of the Myrtaceae family of nutritional and economic importance and wide edaphoclimatic adaptation. For commercial orchard purposes, guava trees are derived from clonal propagation and the production cycle begins with drastic pruning. During its development the guava tree displays varied characteristics that may involve epigenetic mechanisms, such as DNA methylation, revealed by the formation of 5-methylcytosine (5-mC). In order to understand the variability of this mechanism, the %5-mC was evaluated in 22 guava genotypes from a working collection, in an experimental orchard, over five development stages – post-pruning, flowering, fructification, harvest and post-harvest – and subsequently in different tissues. Wide variation in DNA methylation was detected among the genotypes. Hypermethylation was found in one, whereas methylation and demethylation mechanisms were observed in 10 genotypes. Greater variability in DNA methylation among genotypes was found during the vegetative phase, mainly after pruning. On the other hand, uniformity was observed among the genotypes with regards to global DNA methylation during flowering, indicating that this mechanism is conserved in this specific phase. No differences in global DNA methylation was detected in the different plant tissues. In this study, the global epigenetic mechanisms of P. guajava were detected more frequently in vegetative than in reproductive phases. Important Brazilian cultivars such as Paluma, Pedro Sato, Sassaoka and Cortibel SLG exhibit changes in their methylation profiles during their production cycle. Others show greater stability, for instance Século XXI and six Cortibel cultivars, suggesting the possibility of selection for this characteristic. Furthermore, the methylation and demethylation mechanisms of adult guava plants indicate the relevance of epigenetics for the wide edaphoclimatic adaptation of the species. In addition, the clonal propagation of guava may provide an opportunity for the development of this crop through epibreeding approaches.

Genet. Mol. Res. 19(2): GMR18524
DOI: 10.4238/gmr18524
Research Article

Dipteryx alata (Fabaceae), locally known as Baru, is a non-model, native tree species endemic to the Brazilian Savanna (Cerrado), with economic potential due to its use as food, medicine, animal forage, lumber, and in recovery of degraded areas and landscaping. Although D. alata is recognized as an important Brazilian resource, currently there is no genomic information for this species. We generated 22 Gb raw reads from the genomes of D. alata trees using the Illumina MiSeq platform. These were assembled in 275,707 nuclear genomic sequences (N50 = 1598 bp) with a total of 355 Mb, which corresponds to 44% of the whole genome. We detected 21,981 microsatellite regions, of which 49.3% were dinucleotides and 42.7% trinucleotides. We found 421,701 transposable elements (TEs) in 39.29% of the sequences. Long terminal repeat retrotransposons were the most abundant TEs. This is one of the first genomic scale studies for a native Cerrado species. The results can be used for the development of molecular markers for studies on evolution, population genetics and conservation of D. alata.

Genet. Mol. Res. 19(2): GMR18463
DOI: 10.4238/gmr18463
Research Article

Meningiomas are the most common primary tumors of the central nervous system, accounting for 35.5% of cases, considering all age groups. Despite progress made in recent decades, tumorigenesis of meningiomas still remains a challenge. There is a consensus of a need for molecular tools to assist in diagnosis and prognosis of meningiomas. In this context, some studies demonstrate the importance of the role of estrogen and progesterone receptors, as well as the understanding of alterations in microRNA (miRNAs) expression levels in the tumorigenesis of meningiomas. The serum expression profile of miRNAs has been shown to correlate with tumor classification and clinical evolution. Investigation of such miRNAs as biomarkers is of great interest because it would be a non-invasive procedure. We evaluated the tissue and serum expression profile of miRNAs associated with estrogen and progesterone receptor pathways in grade I, II and III meningiomas. Tissue and blood samples from 40 patients with grade I, II and III meningiomas were investigated using real-time PCR to analyze the expression of the miRNAs miR-34a, miR-143, miR-145 and miR-335. The miRNAs miR-34a and miR-145 had significantly lower expression in the tumor tissue samples of grade II meningiomas when compared to grades I and III. We did not observe significant differences in miRNA expression in the blood samples. We concluded that the expression of these miRNAs is not correlated with tumor progression in meningiomas.

 

Genet. Mol. Res. 19(2): GMR18080
DOI: 10.4238/gmr18080
Research Article

Chronic psychological stress alters the hypothalamic–pituitary–adrenal axis (HPA-axis), triggering chronic oxidative-inflammatory states that are associated with physical and psychiatric conditions. However, it is not clear if basal oxidative-inflammatory states triggered by genetic variation affect the HPA-axis by altering cortisol, adrenocorticotropic hormone (ACTH) and dehydroepiandrosterone sulfate (DHEA-S) levels. Humans have a single nucleotide polymorphism (SNP) found in manganese-dependent superoxide dismutase (Val16Ala-SOD2, rs4880), which has two alleles (V and A) which affect the basal efficacy of SOD2 antioxidant enzyme in the mitochondria. The VV-genotype, which presents low SOD2-efficacy, has been associated with chronic inflammatory states, as well as higher risk of depression and self-reported psychological stress. Therefore, basal oxidative imbalance could have some influence on modulation of HPA-axis physiology. We tested this hypothesis comparing morning blood levels of cortisol, ACTH and DHEA-S and other biochemical markers in 90 healthy adult university students previously genotyped for the SOD2-SNP (30 volunteers for each genotype, 26.5 ± 8.7 years old). Only volunteers who self-reported no perception of psychological stress were included in the study. The VV group had higher morning cortisol and ACTH, and lower DHEA-S and brain-derived neurotrophic factor (BDNF) than A-allele subjects. These results indicate some influence of S-imbalance on modulation of this molecule. Therefore, we suggest that genetically controlled pro-oxidative and inflammatory states could modulate physiological markers for stress and neurogenesis.

 

Genet. Mol. Res. 19(2): GMR18586
DOI: 10.4238/gmr18586

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