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Table of contents: 2020

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. 1(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.

Key words:
Genet. Mol. Res. 1(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
Research Article

Genetic variants of SLC2A9 have been reported to be associated with deficiencies in the excretion of urate and with urate transporter gene 9 (GLUT9) activity. However, the evidence remains inconclusive and no information is available about SLC2A9 genetic variants (rs2280205 and rs6820230) association with hyperuricemia (HUA) in the Thai population. We decided to determine whether rs2280205 and rs6820230 alleles of the SLC2A9 gene are associated with HUA in a cross-sectional study that included 193 subjects in urban Bangkok, Thailand. These single-nucleotide polymorphisms were analyzed using unconditional logistic regression. The prevalence of HUA was 31.1%. Males (HUA defined as serum urate levels >7.0 mg/dL) had a higher prevalence of HUA than females (>6.0 mg/dL) (42.7 versus 24.8%). By SLC2A9 SNP association analysis, the rs2280205 and rs6820230 variants were not significantly associated with HUA (adjusted odds ratio (aOR) = 5.59, 95% CI = 0.50-62.95; aOR = 0.77, 95% CI = 0.11-5.45), respectively). The prevalence of HUA was much higher in this urban Thai population than in similar studies in rural areas of the country and also compared to the mean frequency in developed countries such as the USA (usually 10% or less). The haplotypes rs6820230 and rs2280205 did not influence the risk for HUA in these Bangkok subjects. We concluded that the prevalence of HUA is extremely high in urban Bangkok and that these SNPs are not associated with HUA in this population.

Genet. Mol. Res. 19(1): GMR18511
DOI: 10.4238/gmr18511
Research Article

Human papillomavirus (HPV) type 13 is associated with focal epithelial hyperplasia, a benign and self-limited disease of oral mucosa. It is a rare finding, most often seen among young American natives. We carried out a molecular characterization case study of HPV type 13 isolated from a 14-year-old native girl living in Amazonia, in the northern region of Brazil, by describing the E6 and E7 open reading frame (ORF) sequences and comparing them to the only reference sequence in GenBank, which came from a Turkish isolate. The E6 and E7 ORFs were amplified by PCR, and the amplicons were purified and cloned in E. coli using the pCR®-4 TOPO® vector. The purified recombinant plasmids were automatically sequenced, showing 98 and 94% similarity to the E6 and E7 ORFs from the reference sequence, respectively. Six mutations were identified in the E6 ORF, but these changed only three amino acids in the final protein. In the E7 ORF, we found three transversions and one deletion of nine base pairs, which led to three amino acid mutations and three amino acid deletions in the final protein. The mutations in E6 ORF gave no predicted changes in the final protein, while the mutations in E7 were predicted to cause changes in the protein. We conclude that the E6 and E7 ORFs in HPV-13 from the Amazonia and Turkey isolates have a high degree of genetic similarity.

Genet. Mol. Res. 19(1): GMR18393
DOI: 10.4238/gmr18393
Research Article

Nanobodies (Nbs) are mini antibodies 10 times smaller than conventional antibodies. They constitute a new class of antibodies devoid of light chains. Their paratope consists of a single variable domain "VHH" (heavy chain variable domain) formed by a single peptide chain. Nevertheless, Nbs maintain the same repertoire of diversity and affinity characteristics as conventional antibodies. Because of their high specificity, small size, and low production cost, Nbs are emerging progressively as a potential alternative to overcome the limitation of conventional antibodies in targeted therapy. Indeed, the therapeutic spectrum of Nbs has been confirmed in animal models and human clinical trials. The present review provides a summary of the technological advances in the production of Nbs and their potential targeted therapeutic biomarkers over the past 10 years.

Genet. Mol. Res. 19(1): GMR18452
DOI: 10.4238/gmr18452
Research Article

Pigeon droppings carry microorganisms such as Candida spp., which have high genetic variability and are the most common opportunistic pathogens in humans. There is a need to establish fast, reliable and simple laboratory methods to differentiate pathogenic from non-pathogenic yeast species in pigeon droppings, as they can be a source of contamination for humans. We examined yeasts isolated from domestic pigeon droppings through a proteomic approach using matrix-assisted laser desorption ionization (MALDI-TOF). We also examined genetic characteristics of the isolates by restriction fragment length polymorphism (RFLP) PCR and enterobacterial repetitive intergenic consensus sequences PCR methods. Five colonies isolated from domestic pigeon droppings collected at three different sampling locations on the roof of a Brazilian university building were identified as Candida krusei and Candida rugosa by MALDI-TOF. Phylogenetic studies using the two PCR-based molecular methods were able to differentiate the species C. krusei and C. rugosa into different groups. These genomic methods gave similar results and proved useful for genetic diversity analysis and interspecific differentiation of Candida isolates.

Genet. Mol. Res. 19(1): GMR18521
DOI: 10.4238/gmr18521
Research Article

Grapes are an important fruit crop in Kazakhstan. Modern viticulture requires inclusion of traditional morphological and agronomical methods in the development and sustaining of grapevine culture as well as molecular approaches. We used molecular markers and leaf shape analysis to characterize grapevine cultivars developed in Kazakhstan compared with European and Asian varieties. A set of 17 landmarks describing grapevine leaf shape were analyzed using generalized procruste and principal component analyses in order to evaluate shape variation among cultivars. Mean leaf shapes were obtained for 94 cultivars. Outer tip pattern had the most impact on leaf shape variation. Principal component analysis revealed the complex nature of grapevine leaf shape variation and demonstrated the differential impact of leaf landmarks on shape variation. The standard set of six simple sequence repeat markers used for molecular genotyping was sufficient to differentiate each of the cultivars by a distinct allelic profile. Kazakh grapevine cultivars were found to be an admixture of European and Asian varieties. However, for some cultivars, there was no confirmation of identity with respect to pedigree either by bibliography or comparison with the Vitis International Variety Catalogue. Also, 11 markers for resistance-associated alleles in corresponding loci (RUN1, REN1, REN3, Rpv3, Rpv10, and Rpv12) were applied to characterize Kazakhstan and some parent cultivars with different levels of resistance to mildew pathogens.

 

Genet. Mol. Res. 19(1): GMR18482
DOI: 10.4238/gmr18482
Research Article

Plant breeders need to understand the genetic diversity and relationships between genotypes to choose parents of segregating populations. We evaluated the genetic diversity of 35 soybean genotypes and determined their potential as parent lines in breeding programs by analyzing their agronomic traits and microsatellite markers. Phenotypic analysis was carried out in the field at Fazenda Capim Branco, a research station of the Federal University of Uberlândia. Nine microsatellite markers and seven agronomic traits in were evaluated in 35 conventional soybean genotypes. Genotypes were grouped by UPGMA and Tocher cluster analyses, and molecular analysis was used to calculate polymorphism for each microsatellite locus. Nine microsatellite markers amplified 26 alleles, ranging from two to four. Polymorphism varied between 0.29 and 0.66, and averaged 0.44. The number of days to maturity was the characteristic that contributed the most to yield (29.44%). Seven potential parents (G11, G12, G16, G21, G22, G26 and G33) had average grain yields higher than 5000 kg ha-1. UPGMA and Tocher methods identified groups from the matrix of phenotypic and molecular data. Hybrids of G11, G12, G16, G22, G26 and G33 genotypes segregated promising populations with superior genetic variability.

Genet. Mol. Res. 19(1): GMR18105
DOI: 10.4238/gmr18105
Research Article

Signal transducers and activators of transcription (STAT5B), dopamine receptors D2 (DRD2), bone morphogenetic protein (BMP15), and melatonin receptors type C (MTNR1C) play an important role in growth and reproductive traits in chickens. We studied the 9th generation of 93 Pradu Hang Dam and 95 Chee chickens examining PCR-restriction fragment length polymorphisms to genotype for growth and reproductive traits. There was no significant association between other traits, but we confirmed that in Pradu Hang Dam, DRD2 was associated with egg weight at the age at first egg while BMP15 was associated with egg weight at 300 days and body weight at 300 days. Moreover, MTNR1C was associated with egg weight at 300 days and egg number at 300 days. In Chee, MTNR1C was associated with egg number at 300 days. We suggest that DRD2, BMP15, and MTNR1C be used to select for early maturity and growth, while MTNR1C be used for egg number in Pradu Hang Dam. In Chee, MTNR1C can be a marker to improve egg production. For improving the egg production in Thai native chickens, we recommend that MTNR1C be used in marker-assisted selection.

Genet. Mol. Res. 19(1): GMR18504
DOI: 10.4238/gmr18504
Research Article

In this study, a modified CTAB method combined with sodium sulphite and silica particles for the extraction of high-quality genomic DNA of recalcitrant plants such as malva, coffee, avocado, mango, olive, jatropha and tillandsia is reported. Maceration with liquid nitrogen, phenol treatment, RNase digestion and the ethanol precipitation step are eliminated using this methodology. The quality of the isolated DNA permits downstream molecular applications including AFLP, restriction, ligation and PCR amplification and sequencing of plant barcode genes (matK and rbcL). The cost per sample (about US$ 0.14) is quite low compared to any commercially available kit. This methodology is reproducible and could be used for a broad spectrum of woody plants with a high content of polyphenols and polysaccharides compounds, without the need for a commercial kit or expensive reagents, providing a viable alternative for laboratories with low budgets.

Genet. Mol. Res. 19(1): GMR18510
DOI: 10.4238/gmr18510
Research Article
Authors:

Various approaches use gene trees to infer species trees produced from incomplete lineage sorting. Generally, one of these approaches is used to deduce the rooted species tree from a rooted gene tree, or another method can be used to determine the unrooted species tree from an unrooted gene tree. Typically, this unrooted species is then rooted through at least one outgroup. However, in theory, the unrooted gene tree can be used consistently and directly to infer the rooted species tree without using an outgroup. We used an unrooted gene tree with the assumption of a multispecies coalescent model to infer the rooted species tree by using the approximate Bayesian computation (ABC). In certain cases, this could be useful, especially when it is hard to locate a fitting outgroup neglected by gene trees. To address the challenges of increasing the taxa number, an ABC was used to gauge the rooted species tree of a large number of taxa, using an unrooted gene tree to develop the rooted species tree. This is the first ABC application that can handle very large numbers of taxa. Based on the results, the Robinson-Foulds (RF) distance is generally equal to 2 when the species tree is in imbalance. When the shape of the species tree is balanced, the RF distance is normally equal to 0. Out of all shapes of species trees, the most recent one is the most appropriate for every clade.

Genet. Mol. Res. 19(1): GMR18433
DOI: 10.4238/gmr18433

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