Current issues

Table of contents: 2020

Research Article

Probiotics are live nonpathogenic microorganisms extensively used in food, pharmaceutical and medicinal industries. Recently, attention has focused on specific features of probiotics and on the abilities of some long known and recently described species of this group. In general, desired features of probiotics include resistance to acid and bile salts to avoid dysbiosis and induction of immune system development. The advent of next-generation sequencing technology has propelled the genomic area, allowing a search for probiotic features in a wide range of probiotic species, especially bacteria. In this context, functional genomics analyses can help interpret big data, correlating the findings with comparative genomics analyses, in a search for direct applications. To select the articles in this review, we used the following indexing terms: (probiotics OR probiosis) AND (genomics OR transcriptomics OR proteomics OR metabolomics OR culturomics) AND bacteria. Proteomics and transcriptomics methodologies reveal important information about proteins and transcripts differentially expressed under specific conditions that mimic host environments in health and disease. In addition, new research approaches have been developed for probiotics, such as metabiotic and metagenomic analyses of host microbiota. Also, we examined probiotic related features, including bacterial safety aspects; tolerance towards digestive constraints, such as gastric juice and bile salts; bacterial pathogen exclusion mechanisms; adhesion-related genes; antimicrobial peptides; immune development and function; omics; metagenomics; culturomics; functional genomics; transcriptomics; proteomics; metabiotics and metabolomics. In summary, currently there is considerable interest in probiotic bacteria, and structural and functional genomics analyses have potential to help research in this area.

Genet. Mol. Res. 19(2): GMR18599
DOI: 10.4238/gmr18599
Research Article

Genetic improvement of soybean involves various biometric techniques and genomic tools. Controlled hybridization is an indispensable tool of the researcher; however, considering the complexity of the process, floral sensitivity and a strong effect of environmental conditions, the procedure is difficult to control. The objective of this work was to define the optimal periods for artificial hybridization of soybean, in order to maximize the number of seeds formed, and to understand the relationships between the agronomic characteristics of the parents and progenies. The study was carried out in Campos Borges, Rio Grande do Sul, Brazil, during the agricultural years of 2013, 2014, 2015, 2016, 2017 and 2018. The crosses were made during the initial period of flowering of the culture in the greenhouse, totaling 498 directed crosses. There was considerable variability in the number of crosses successfully made, in seed number and viability, and in the crossing index. The efficiency of obtaining heterozygous soybean seeds was 13.8% using planned crosses under controlled conditions. The optimum period to maximize the number of seeds was from 8:15 to 9:45 AM, the number of seeds being inversely proportional to the advance in the day hours. The flower color and the growth habit of the parents express a similar tendency as the time of the cross is determinant for the success of this stage of breeding.

Genet. Mol. Res. 19(2): GMR18181
DOI: 10.4238/gmr18181
Research Article

Cluster of differentiation 1 (CD1) is antigen-presenting molecule involved in the presentation of lipid and glycolipid antigens to specific T cells and natural killer T cells. They exhibit a low rate of polymorphism compared to classical major histocompatibility complex presenting molecules. In this case control study, we examined a possible association of genetic polymorphism in CD1A and CD1D genes with colorectal cancer (CRC) in a Saudi population consisting of 120 patients with CRC and 118 matched controls. They were genotyped for the CD1A T/C 622 and CD1D A/T 354 polymorphisms using PCR-SSP. An apparent protective effect of the C allele, heterozygous TC and additive TC + CC genotypes against CRC was observed. These associations were found only in individuals >57 years.   No association was observed between the CD1D A/T 354 polymorphism and CRC.

Genet. Mol. Res. 19(2): GMR18563
DOI: 10.4238/gmr18563
Research Article

Iron deficiency anemia mostly affects children and adolescents, women of childbearing age and pregnant women due to their high iron requirements. The MTHFR enzyme helps maintain the pool of folate and methionine, avoiding the accumulation of homocysteine. Patients with elevated levels of homocysteine have a wide range of clinical features, including iron anemia, because cysteine is a protein derived from the metabolism of homocysteine and is related to iron homeostasis. The objective of our research was to determine whether the MTHFR gene A1298C polymorphism is related to the development of anemia in patients with iron deficiency anemia. We analyzed genetic polymorphisms using blood samples from blood donors who were part of the control group as well as patients with iron deficiency anemia through PCR and electrophoresis techniques. Among 42 patients, 21 had the AC genotype, 5 the CC genotype and 16 the normal AA genotype. In the control group, 58 blood donor samples were analyzed, of which 44 showed the genotype (AA) and 14 were identified as having the heterozygous (AC) genotype; no samples with the CC genotype were identified. Thus, we found a frequency of 0.62 and 0.24 of the A1298C polymorphism of the MTHFR gene for the patient group and the control group, respectively, which is a significant difference. We conclude that the A1298C polymorphism of the MTHFR gene may contribute to the development of iron deficiency anemia.

Genet. Mol. Res. 19(2): GMR18522
DOI: 10.4238/gmr18522
Research Article

Camu – camu, Myrciaria dubia (Myrtaceae) is a native species of the Amazon Rainforest that has been attracting attention worldwide and arousing great interest in the food and pharmacological industries due to the high concentrations of ascorbic acid in its fruit, which is exported to several countries. Characterizing different materials of M. dubia by means of molecular markers allows integration of agronomic and molecular information to aid in the search for more promising varieties. We examined the genetic variability of 11 populations of this species distributed along the Branco River hydrographic basin in state of Roraima in northern Brazil. The populations were defined taking into account the origin of the subsample. The 55 sub-samples present in the Embrapa Roraima Germplasm Collection were evaluated using five ISSR initiators (UBC 811, UBC 812, UBC 817, UBC 868 and UBC 880). The five primers tested generated 64 fragments, with a 98% polymorphism rate. The greatest genetic variation was expressed within the populations (66.6%), while the lowest divergence was determined among the populations (33.4%) of the collection. There was a significant correlation between genetic and geographical distances (Mantel test, r = 0.3%, P < 0.01). Analysis with the UPGMA method gave four subgroups showing that various individuals are genetically divergent and can be used in genetic breeding programs.

Genet. Mol. Res. 19(2): GMR18418
DOI: 10.4238/gmr18418
Research Article

The soybean cultivars currently available for cultivation are used mainly due to their productive and phytosanitary characteristics, without defining specific centesimal compositions as an ideotype.  Our study aimed to contribute to this aspect and to identify profiles and establish similarities between the genotypes, taking into account components such as starch, lipids and mineral materials in 54 soybean genotypes. The experiment was conducted in the 2017/2018 agricultural year in Campos Borges, RS. The treatments consisted of four cultivars, considered pure lineages, and 50 F5 segregating families (theoretical level of inbreeding of 94% with approximately 6% heterozygosis). The experimental units were composed of a sowing line five meters in length, spaced 0.45 m. The characters of agronomic interest measured in the grains collected from 10 random plants were: protein, humidity, lipids, total fibers, mineral material and starch. The percentages of the characters of agronomic interest: proteins, lipids, fibers, mineral matter, starch and oil were determined in intact soybeans by the technique of reflectance in the near infrared. There was genetic dissimilarity among the 54 soybean genotypes. The protein character was the most decisive factor for genetic variability, based on Singh's relative contribution. Using patterns such as standardized Euclidean mean distance, Tocher's optimized method and the UPGMA clustering method, it was possible to show patterns in the genotype clusters. Based on these results, we can define crosses between accessions with genetic potential for the formation of genotypes with superior centesimal characteristics.

Genet. Mol. Res. 19(2): GMR18423
DOI: 10.4238/gmr18423
Research Article

There is little information available concerning the effects of different sources and levels of phosphorus in the soil on lettuce quality and production. To help determine an optimal strategy, we used complete randomized blocks, in a 2x3x5 factorial experimental design, with two commercial lettuce cultivars widely used in Brazil (Americana (iceberg lettuce) cv. Lucy Brown and Crespa (leafy lettuce) cv. Vanda) fertilized with one of  three phosphate sources [MAP, Polyblen or Triple Super Phosphate (SPT)], at five concentrations (0, 250, 500, 750 or 1000 kg of P2O5 ha-1), with three replicates planted on a farm in Mineiros, GO. The variables stem height, head diameter, number of commercial and non-commercial heads and commercial fresh weight, were analyzed at 40 days after planting. There was a triple interaction for these factors, when their averages were broken down to qualitative and quantitative effects. The variables were correlated, and affinities with the genotype x phosphate source interaction were found. Based on the yield data, the recommendations for Americana are 911, 680 or 457 kg P2O5 ha-1 from MAP, Polyblen, and SPT, respectively. For Crespa the recommendations are 580 or 611 kg P2O5 ha-1 from MAP and SPT, respectively.

Genet. Mol. Res. 19(2): GMR18424
DOI: 10.4238/gmr18424
Research Article

Nitrogen (N) is the most important soil nutrient for common beans; the main sources are mineral N fertilizers and symbiotic N fixation (SNF). The common bean (Phaseolus vulgaris) is a N fixing species, but breeding programs have neglected this fact. We investigates black seeded elite lines grown with mineral N fertilization or using inoculation with rhizobia to select lines with broad adaptability, high yield stability, and high agronomic performance. Fifteen black seeded common bean elite lines and cultivars, all of them selected under mineral N fertilization, were evaluated in major bean crop regions in the Brazilian states of Goiás, Paraná and Distrito Federal in different growing seasons and years, totaling 13 environments (combination of location/growing season/year). Two side-by-side experiments were set up in each environment, one exclusively with mineral N fertilization (total of 80 kg ha-1 of N) and another with Rhizobium inoculation only. The agronomic traits evaluated were seed yield, 100-seed weight, sieve yield, reaction to anthracnose and angular leaf spot. The nodulation traits evaluated were number of nodules and nodule dry matter, specific weight of nodules and relative nodulation index. The N source significantly affected seed yield, 100-seed weight and reaction to anthracnose. All lines produced a higher yield and had higher 100-seed weight under N mineral fertilization; but they were more resistant to disease when inoculated with rhizobia. The interaction between lines and N sources was significant for seed yield, sieve yield, and reaction to anthracnose. However, it did not affect selection of the best lines regardless of N source. The cultivars BRS FP403 and BRS Esteio are recommended for planting with either of the two N sources because they were the highest yielding and exhibited high adaptability and stability under both N supply systems. Selection of superior lines does not appear to depend on the source of N used during crop development. The lines BRS Campeiro and CNFP 15177 gave the highest nodulation index, indicating that these lines should be used in crosses with high yielding lines to produce lines with high seed yield and high nodulation, reducing possible yield losses in SNF systems compared to those that useonly mineral N fertilizer.

Genet. Mol. Res. 19(2): GMR18491
DOI: 10.4238/gmr18491
Research Article

 Malagueta (Capsicum frutescens) is one of the most widely consumed and cultivated Brazilian hot peppers. It is an important crop for smallholder farmers throughout the country. Currently, the demand for new hot pepper cultivars is increasing. A germplasm collection of C. frutescens is maintained at Embrapa Vegetables, Brasilia, Brazil, the branch for vegetable crops of the Brazilian Agricultural Research Corporation (EMBRAPA). This is considered to be the main collection representing the variability of this species in the country. Four core collections of 13 accessions each were established through 1) random selection, 2) selection based on morphological and 3)SSR similarity groups and 4) selection based on SSR similarity groups associated with virus incidence. Characterizing the original germplasm collection (103 accessions) through 57 morphological characters, 239 alleles of 24 microsatellite (SSR) loci and incidence of six virus species provided the information used for selecting the accessions. Discriminating C. chinense and C. frutescens species proved to be inaccurate when relying only on morphological characterization for 5% of the accessions, whereas molecular characterization was decisive for the species identification of all accessions. The SSR allelic variability within each core collection was compared with the full C. frutescens collection. Selection based on SSR grouping associated with data on viruses incidence provided the highest allelic representativeness among the four strategies (77% of the allelic variability present in the full collection), in addition to satisfactorily representing the Brazilian geographic diversity. The core collection based on morphological characters was also highly representative of the allelic variability (73%) in the original full collection.

Genet. Mol. Res. 19(1): GMR18503
DOI: 10.4238/gmr18503
Research Article

Since 2012, CRISPR/Cas technologies have revolutionized the field of modern molecular biology and biotechnology due to their ability to direct a Cas nuclease to cleave specific nucleic acid sequences with a guide RNA (crRNA), providing dynamic tools for many applications, being naturally derived from the adaptive immune system of prokaryotes. The most widely used toolbox for genome editing, modulation, and detection contains types II, V and VI of class 2 systems, categorized and characterized by Cas9, Cas12, and Cas13, respectively. In recent years, given the wide application of Class 2 CRISPR-Cas systems as molecular tools, there has been a focus on the discovery, characterization, and specificity of Cas effector nucleases from these systems, generating even more possibilities for the development of new applications. We reviewed the scientific literature on current knowledge of CRISPR-Cas systems and recent advances in biotechnology involving class 2 systems between 2000 and 2019. For this, we searched for original articles indexed in the following databases: PubMed, Scopus, SciELO, Periodicos Capes and Google Scholar, and used keyword combinations  such as “CRISPR / Cas Class 2 systems”, “Cas9”, “Cas12”, “Cas13”, “Genome editing” and “diagnostic detection”. Eighty original articles were obtained, 55 of them were selected for this review because they match with the keywords and the years of publication.

Genet. Mol. Res. 19(1): GMR18478
DOI: 10.4238/gmr18478
Research Article

The aim of this study was to develop microsatellite markers for Ololygon centralis, an endemic tree frog species of the Cerrado biome of Brazil, to improve population genetics studies with focus on conservation and ecology. Sets of primers were designed from sequences derived from high throughput sequencing (Illumina-Miseq). Five polymorphic microsatellite loci were characterized for 30 individuals from three populations of the state of Goiás, Brazil. All loci combined presented a probability of identity (I) equal to 1.13x10-6 and paternity exclusion (Q) of 0.993. The number of alleles per locus ranged from 5 to 17, with a mean of 10 alleles. The expected and observed heterozygosity ranged from 0.41 to 0.98 and from 0 to 1, respectively. The global fixation index Fst found was 0.213 (P < 0.05). These markers will be useful for genetic diversity analysis and will contribute to population genetics studies for species of the Scinax (=Ololygon) catharinae clade.

Genet. Mol. Res. 19(1): GMR18528
DOI: 10.4238/gmr18528
Research Article

The genus Atelopus is one of the most diverse of the Bufonidae family; because of their bright color, they are referred to as harlequin frogs. They occur in mature tropical forest areas and in this region, these forests are under anthropic pressure and limited to fragments, which facilitates the action of pathogenic fungi. One of these toad species, Atelopus hoogmoedi, is only found to the north and south of the Amazon River. Based on genetic data this species name represents more than one evolutionary unit. To explore this premise, we compared individuals of A. hoogmoedi collected south and north of the Amazon river in the state of Pará, Brazil. The DNA was extracted by the phenol chloroform method from eight individuals, seven from Monte Alegre (north of Amazon River) and one from Anapu (south of Amazon River) and then amplified via PCR using a mitochondrial 16S rRNA marker. Phylogenetic analysis of maximum likelihood for A. hoogmoedi revealed a paraphyletic group with three lines: French Guiana 1 and 2, Guyana and Monte Alegre, and Anapu. The genetic distance between Anapu and Monte Alegre was 2.9%. According to the Automatic Barcode Gap Discovery in both Simple Distance and Kimura 2 Parameters models, A. hoogmoedi collected in Anapu is recognized as a distinct species from those of the Guiana Shield. Thus A. hoogmoedi to the south of the Amazon River was classified as an unconfirmed candidate species, requiring more collections and access to individuals from other localities of its occurrence for confirmation.

Genet. Mol. Res. 19(1): GMR18392
DOI: 10.4238/gmr18392
Research Report

Tomatoes are an important crop in Brazil. In advanced stages of breeding programs, the capacity to select or reject tomato lines becomes complex. We selected tomato lines comparing different selection strategies. We evaluated 115 advanced tomato lines from the Tomato Germplasm bank of the Federal University of Uberlândia. The following characteristics were evaluated: total chlorophyll content, foliar temperature, number of fruits, yield, distance between the first cluster and the soil, transverse and longitudinal fruit diameter, internode length and total soluble solids. Data was submitted to variance analysis, Scott-Knott's test of means, and selection gains estimates: direct and indirect selection, the classic Smith and Hazel index (SH), the Williams’ base index (W), the genotype-ideotype distance index (GID) and the Mulamba and Mock’s sum of ranks (MM). According to the Scott-Knott's test, the lines clustered in two groups for the variables number of fruits, yield, and distance between the first cluster and the soil. There was no consistency between the different selection indexes. Selecting 9% of the lines, total selection gains estimate for selection indexes were 12.57 (SH), and 38.57% (MM). With genetic gains more equally distributed among the characters, the MM index is suggested as the most appropriate for advanced stages of tomato breeding programs.

Genet. Mol. Res. 1(1): GMR18462
DOI: 10.4238/gmr18462
Research Article

Sex differentiation in males is a highly complex phenomenon based on synergistic interactions between genes and their coded proteins. The Sox9 gene plays a critical role in embryonic development, cell lineages, and stem cell maintenance during testicular development. Sox9 gene expression is associated with sex reversal, with a 46, XX karyotype in males and regulating normal functioning of Sertoli cells during spermatogenesis. Clinically diagnosed infertile males (n = 40) with non-obstructive azoospermia (NOA) with respective controls (n = 38) were included in the study. Blood samples were collected and after isolation of genomic DNA, the expression of the Sox9 gene (DNA copy number variations) was measured by fluorescence based quantitative PCR, deletion of AZFc locus determined by STS markers, karyotype analysis and SRY using GTG banding and FISH techniques, respectively. Cytogenetics findings revealed that more that 40% of cases had mosaicism with 46, XY/47, XYY karyotypes and a high frequency of deletion of AZFc (>70%) regions as compared to AZFb (>20%) using STS markers in the cases of NOA. The mean relative fluorescence values of Sox9 gene expression differed significantly between cases (0.32) and controls (0.87) (P < 0.001) with a confidence interval at 95% of 0.0310 - 0.787 in the cases of NOA. SRY + ve cases had higher Rfv of Sox9 gene expression when compared to SRY- ve cases. There was a positive correlation between stem cell Sox9 gene expression, mosaicism and AZFc region affecting a non-disjunction event during spermatogenesis and fertility.

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Genet. Mol. Res. 19(1): GMR18464
DOI: 10.4238/gmr18464
Research Article

Surgery, chemotherapy and radiotherapy, which are known as conventional cancer treatments, have been applied for more than 40 years. Because the target of chemotherapy is tumor cells, treatment agents are used at high doses during application and this creates toxic responses, causing side effects in normal cells. Furthermore, due to the high doses, cancer cells develop resistance to the agent and, as a result, success of the treatment gradually decreases. That is why chemotherapy is combined with various other cancer treatments to increase the therapeutic efficacy while reducing the side effects. In this study, the cytotoxic effect of endostatin, vinorelbine and their combination on breast cancer cell line MDAMB 231 was researched in vitro. In addition, the effects caused by the combination on TRAIL, TNF-α and caspase-2, 3, 6, 8 and -9 proteins, which undertake key roles in apoptosis were investigated. Cytotoxic effects of both agents and their combinations on normal epithelial cells (293 T) were also studied and compared. We found that endostatin is cytotoxic for cancer cells through caspase-8 and TNF-α activation, which affects the extrinsic pathway of apoptosis Combining endostatin as an anti-angiogenic agent with vinorelbine significantly suppressed the cytotoxicity that vinorelbine exerts on normal cells.

Genet. Mol. Res. 1(1): GMR18497
DOI: 10.4238/gmr18497