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Table of contents: 2022

Research Article

The dehydration responsive element (DRE)-binding proteins (DREB) play a role in the signaling network that activates many abiotic stress-responsive genes. We isolated and molecularly characterized the DREB gene from ancestral diploid wheat species growing in Azerbaijan and native to this region. This territory is included in a region that is considered the center of origin of cultivated wheat. One-week-old seedlings of Triticum urartu (Au), Aegilops speltoides (B) and Ae. tauschii (D) were used for genomic DNA extraction. Gene-specific primer pairs were applied forisolation of the DREB gene. The amplification products were purified using a gel extraction kit and sequenced. Data analysis was performed using FGENESH, BLAST, INTERPROSCAN, SMART, MAFFT, ExPASy, ProteinPredict, and PSIPRED tools. Ensembl Plants and NCBI were used as integrative resources. Numerous SNPs and nine microindels were detected in the partial target sequence of the DREB gene in Ae. speltoides. Nonsynonymous SNPs were determined in T. urartu (1 transition and 5 transversions), and Ae. tauschii (2 transitions and 2 transversions). Analysis of amino acid sequences encoded by the putative DREB genes revealed a conserved AP2/ERF domain, with two conserved functional amino acids (14th valine and 19th glutamic acid) that play crucial roles in the recognition of the DNA-binding sequence and two tryptophan rings that determine the geometry of the GCC-box binding domain. Nuclear localization signal and conserved Ser/Thr-rich region were observed in the corresponding amino acid sequences. One α-helix and three β-sheets were detected in the secondary structure of the AP2 domain. The isolated sequences of DREB gene from T. urartu and Ae. tauschii were confirmed and registered in NCBI with Accession Numbers MZ935739 and MZ935740. Identification of the DREB gene in wheat progenitors and its characterization is important for evaluating their genomic material for possible use to enhance the diversity of wheat cultivated under stress conditions.

Genet. Mol. Res. 21(3): GMR19035
DOI: 10.4238/gmr19035
Research Article

We developed a new and simple feeding device for Drosophila melanogaster. In addition, we tested three negative geotaxis methods (measuring the percentage of the flies able to climb 8 cm in 8 s, measuring the distance climbed in 3 s, and measuring the distance climbed in 8 s). The flies were exposed to chlorpyrifos (CPF) using the new feeding device. Our results demonstrated that the three methods for measuring negative geotaxis could be used interchangeably with respect to the needs and conditions of the experiments; however, we recommend the 8 s method with PAST software because the other two methods were carried out using manual measurements. The use of this free software makes the process more accurate with no additional cost. We found that CPF caused impairment in locomotor activity, reduction in AChE activity, and disturbance of the dopaminergic pathways in D. melanogaster, suggesting that CPF toxicity is not confined to the cholinergic system. This study provides a new system to study neurodegenerative damage using a user-friendly and no-cost software for measuring climbing activity in D. melanogaster.

Genet. Mol. Res. 21(3): GMR19056
DOI: 10.4238/gmr19056
Research Article

Research on genes affecting phenotypic variation in milk production and composition from indicine (Bos indicus) cattle is imperative, since these breeds are important tropical genetic resources, and there have been few studies investigating the genetic basis of these traits. We identified polymorphisms in k-casein (CSN3), b-lactoglobulin (LGB), thyroglobulin (TG) and prolactin (PRL) and examined their effect on milk and composition traits in the Guzerá breed. DNA samples of 260 Guzerá  cattle selected for dual purpose use were genotyped. Allele frequencies observed for the A allele were 0.83, 0.18 and 0.25 respectively for CSN3, LGB and PRL genes, while for the TG gene T allele had an allele frequency of 0.09. For all polymorphisms evaluated, observed genotypic frequencies were in agreement with those expected according to the Hardy-Weinberg Equilibrium hypothesis. A polymorphism association study evaluated breeding values (BV) for 305-day milk (BV-M), fat (BV-F), and protein (BV-P) production, employing the allele substitution model using a sample of 139 cows belonged to 27 full and half-sib families of a MOET (multiple ovulation and embryo transfer) selection nucleus. Association was found between the LGB polymorphism and BV-M, BV-F and BV-P. Animals with LGB AA genotype have, on average, higher BV when compared to animals with LGB AB and BB genotypes (277.85 kg for BV-M, 12.09 kg for BV-F and 9.33 kg for BV-P).  These findings contribute to a better understanding on the influence of these polymorphisms on milk production traits in Guzerá cattle.

Genet. Mol. Res. 21(3): GMR19046
DOI: 10.4238/gmr19046
Research Article

The Brazilian Testudinidae family is widespread across South America. It includes Chelonoidis denticulatus, the largest tortoise in South America and Chelonoidis carbonarius, found mostly in the north and northwestern part of the continent. Using hemoglobin to identify species is cheaper than other methods such as DNA sequencing and can offer useful information, since the hemoglobin molecule is a well-preserved protein chain during the evolution of species. Thus, in order to establish a hemoglobin profile for the Brazilian Testudinidae C. denticulatus, C. carbonarius and morphotype 1, hemoglobin electrophoresis was performed at acid pH in phosphate agar and at alkaline pH in cellulose acetate, in order to visualize the specific fractions of each species. High performance liquid chromatography was used for the quantification of fractions. For an in-depth analysis and better detailing of the hemoglobin profile of the species, polypeptide chain electrophoresis was performed at acid and alkaline pH. We observed differences in the hemoglobin profiles of C. denticulatus in relation to C. carbonarius and morphotype 1, which suggests that this methodology, not common in taxonomic studies, can help determine relationships between species, since hemoglobins are proteins with well-preserved genes. We found differences in hemoglobin mobility between C. denticulatus, C. carbonarius and morphotype 1 in electrophoresis at alkaline pH, however, the behavior of globin chains was similar between the three groups. High performance liquid chromatography showed different retention times in the globin fractions of C. denticulatus and C. carbonarius, but not between C. carbonarius and morphotype 1, indicating that, possibly, the divergence time between C. carbonarius and morphotype 1 is more recent than the divergence between C. denticulatus and C. carbonarius, due to the highly conserved character of this functional protein. Thus, considering the high degree of conservation of hemoglobins in vertebrates, and the differences observed in electrophoresis at alkaline pH and HPLC, we infer that C. carbonarius and morphotype 1 present a common branch.

Genet. Mol. Res. 21(3): GMR18977
DOI: 10.4238/gmr18977
Research Article

Quantitative PCR puts great demands on DNA quality and relies on a comparison of fragment amplification between two chromosomes using different primers. The use of a single primer pair capable of reliable relative comparative amplification would be a great advantage.  Using this approach, we developed a rapid, high-throughput, semi-automated and cost-efficient multiplexed method for molecular determination of chromosomal sex called MQS-PCR – multiplex quantitative sexing PCR. DNA sequences located on different chromosomes and differing in length can be amplified and fluorescently labelled with a common fluorescent primer and conveniently separated and detected using capillary electrophoresis. In this method, the intensity of amplification of each of the fragments is compared to determine DNA dosage. MQS-PCR achieves 100% analytical sensitivity and specificity in detecting normal and abnormal sex chromosomal complements (45,X, 46,X,i(X)(q10), 46,X,i(X)(p10), 46,X,X(p-), 46,X,X(q-), 47,XXX, 47,XXY and 47,XYY). It is a reliable, low cost, and rapid detection method for the determination of chromosomal sex and sex chromosomal abnormalities in human samples.

Genet. Mol. Res. 21(3): GMR19067
DOI: 10.4238/gmr19067
Research Article

Wheat is considered a basic cereal for civilization with great economic importance for world agriculture. Currently, wheat is planted in the south, southeast and midwest regions of Brazil. Brazilian consumption of wheat will grow in the coming years due to population increase, which will require developing new cultivars for non-tradtional regions. We investigated commercial wheat genotypes grown at various sowing dates in a region with high temperature conditions. The experiment was conducted during the two summer and two autumn seasons. Sowings were March 10, March 20, April 1 and April 10. The agronomic traits (grain yield, plant height, spike size, total spikelets per spike and fertile spikelets per spike) for four commercial genotypes were evaluated. The experimental design was randomized blocks in a factorial scheme, corresponding to four genotypes, four sowing seasons and two years of cultivation. The second sowing year gave the best performance for the genotypes. Genotypes BR 18 and BRS 404 gave superior agronomic performance, standing out in the third and fourth sowing dates, under these culture conditions.

Genet. Mol. Res. 21(2): GMR18993
DOI: 10.4238/gmr18993
Research Article

Genome mapping is a simplified representation of molecular markers or nucleotide sequences in chromosomes; developing accurate and dense maps is crucial for marker-assisted selection. We developed and compared genetic linkage maps obtained using JoinMap 4.0 and GACD with a physical map obtained using BLAST analysis based on Eucalyptus SNPs transferred to guava, Psidium guajava (Myrtaceae) - to serve as a reference for trait mapping in this crop. Genotyping was conducted on 112 individuals from an experimental cross between a well-known commercial cultivar and an exotic genotype (Pedro Sato × Purple guava), using the Euchip60K SNP chip, version 2.0 (72,202 SNPs); 79% of the SNPs were monomorphic. After data filtering, 1120 markers were used for map construction. The JoinMap 4.0 linkage map had 203 markers, spanning 1405.2 cM, with an average marker distance of 7.7 cM. The GACD linkage map had 186 markers and spanned 1392.7 cM, with an average marker distance of 8.8 cM. JoinMap and GACD disagreed on the estimated distances and SNP ordering. GACD showed a greater limitation than JoinMap 4.0 as it ordered markers according to their parental origin. The physical map developed using BLAST consisted of 694 hits (e-values from 8xE-10 to 1.15xE-26), spanning 434.88 Mb, with an average marker interval of 0.62 Mb. Both linkage maps showed linkage groups with segments from several chromosomes compared to the physical map, indicating limitations. These results highlight the effectiveness of physical mapping through BLAST to overcome linkage mapping limitations, such as in marker grouping and ordering. The physical maps proposed here can serve as a reference for mapping and QTL estimates in guava.

Genet. Mol. Res. 21(2): GMR19033
DOI: 10.4238/gmr19033
Research Article

X chromosome inactivation (XCI) compensates for the imbalance in gene expression between sexes. In mice, it is well established that the long non-coding RNA (lncRNA) X-inactive specific transcript (XIST) is essential for initiating XCI. The most well-known antisense transcript of the mouse XIST locus is TSIX, a negative modulator of XIST. However, in cattle, these events are not yet well established. In this study, we characterized the patterns of strand-specific transcription along the XIST locus in bovine fetal placenta, since understanding the regulation of gene expression in the placenta and of the transcripts involved in XCI in order to minimize embryonic and fetal losses due to the use of assisted reproduction techniques (ARTs) is key for livestock production. Sense transcription was detected throughout the XIST locus in male and female, and antisense transcription was detected in exon 1 of female fetal cotyledons. The sense transcripts may be lncRNA XIST, while the antisense transcript identified in exon 1 is not TSIX, but rather other uncharacterized RNAs. Moreover, our results show the relevance of taking into account the possibility of antisense expression for gene expression studies, especially in non-coding RNA or pseudogenes loci, where transcription from the two DNA strands is not rare. Taken together, the results show the importance of characterization as an aid to a better understanding of XCI in cattle, considering that epigenetic reprogramming can be impaired in cattle by the use of ARTs.

Genet. Mol. Res. 21(2): GMR19039
DOI: 10.4238/gmr19039
Research Article

This study aimed to examine the effects of curcumin, a phytochemical antioxidant, on the treatment and care of diabetic nephropathy and to contribute to alternative treatment strategies for diabetes. Male Wistar albino rats (8–10 weeks old) were divided into five groups of seven. Experimental diabetes was induced in all rats except for those in Group 1 (placebo group) by administration of 110 mg/kg nicotinamide, followed by intraperitoneal administration (after 15 min) of 55 mg/kg streptozotocin. Groups 1, 3, 4, and 5 were treated with 0.1 ml normal saline (0.9% NaCl), 150mg/kg/day metformin, 10 mg/kg/day glycazide (diamicron), and 200 mg/kg/day curcumin, respectively. Group 2 did not receive any treatment. Kidney tissues of rats were collected for histopathological examination.  There were no significant differences in the kidney dimensions of the rats. In the histopathological evaluation of kidney tissues with diabetic nephropathy, glomerular congestion and destruction were observed. Rats treated with curcumin had significantly less kidney damage, based on histopathological analysis, than those treated with the diabetes drugs.  We conclude that curcumin has protective effects in kidneys due to its antioxidant properties. It has potential for use, in addition to antidiabetic drugs, for diabetes treatment.

Genet. Mol. Res. 21(2): GMR19026
DOI: dx.doi.org/10.4238/gmr19026
Research Article

We investigated single nucleotide polymorphisms of four genes (calpain (CAPN-9 and CAPN-14), calpastatin – CAST, diacylglycerol acyltransferase – DGAT, and leptin - LEP) that are related to beef quality, and we examined changes in the synthesis of proteins that they encode in Nellore cattle. Samples from 95 adult males of commercial origin were analyzed. Genomic DNA was extracted from the longissimus dorsi (sirloin) muscle tissue, identified with PCR-single-stranded conformation polymorphism analysis. For each different pattern identified, the products were sent for sequencing and analyzed using sequence scanner software. The data were analyzed by determining the absolute and relative frequencies of the polymorphisms identified in each gene. For the CAPN-9 gene, sequencing showed five polymorphisms (G/A, T/A, T/C, T/C, and A/G), of which two involved amino acid substitutions (c.5861G>A and c.5498A>G). Sequencing of the CAPN-14 gene revealed four polymorphisms (A/C, G/A, T/C, and C/G), with two involving amino acid substitutions (c.11054 T>C and c.11161C>G). Sequencing of the CAST-5 gene revealed five polymorphisms (C/T, T/C, C/A, C/A, and G/T), four of which involved amino acid substitutions (c.29919C>T, c.29963A>C, c.29978C>A, and c.30019G>T). Sequencing of the DGAT gene revealed six polymorphisms (T/A, G/A, A/T, G/C, A/G, and G/A), four of which involved amino acid substitutions (c.11730A>T, c.11809G>C, c.11858A>G, and c.11927G>A). Sequencing of the LEP gene revealed three polymorphisms (C/T, C/T and T/C), with one involving an amino acid substitution (c.14962T>G). These genes   had a large number of polymorphisms resulting in amino acid differences in Nellore Cattle. These polymorphisms involving amino acid changes may promote functional changes in beef characteristics of Nellore cattle given that these genes are associated with beef quality parameters.

Genet. Mol. Res. 21(2): GMR18983
DOI: 10.4238/gmr18983
Research Article

Cynodon dactylon (Bermudagrass) ise used for forage in Brazilian pastures, as well as in lawns, parks, and sports fields. However, in sugarcane fields, it is a difficult to control weed due to its rapid adaptation and growth mechanisms. It competes with sugarcane plants negatively affecting crop yield. Bermudagrass has polyploidy and easy hybridization, which promote high polymorphism, generating varying responses to the environment. Consequently, understanding the structure and variability of Bermudagrass becomes important for the development of strategies for its management as a weed. We examined the levels of genetic variability and structure of five populations (SP1 to SP5) of Bermudagrass, collected from sugarcane fields in the state of São Paulo (Brazil). Thirteen microsatellites were used. PCoA demonstrated STRUCTURE results (K=4) showing a mixture of SP1 and SP3 populations, with SP2, SP4 and SP5 being the most distant. DAPC also confirmed low genetic differentiation for SP1 and SP3. Genetic variability was found to be greater among than within populations, due to the predominance of vegetative growth of the species, which promotes low diversity, and due to geographic distances, which reduce gene flow or even make it unfeasible. The SSRs showed high resolution in characterizing the genetic diversity and structure of the five populations of Bermudagrass. The findings of this study may help to establish biological control methods against Bermudagrass in sugarcane fields.

Genet. Mol. Res. 21(1): GMR19010
DOI: 10.4238/gmr19010
Research Article

The SARS-CoV-2 pandemic has demonstrated the need for genomic epidemiology surveillance. To date, various methodologies have been applied, including metagenomic approaches and amplicon-based sequencing associated with high-throughput sequencing platforms. We adapted some details in amplicon-based sequencing using a SARS-CoV-2 community panel (Illumina AmpliSeq), with additional modifications for balanced and high-quality sequencing using the MiSeq platform. The modified protocol was used to detect circulating SARS-CoV-2 variants in Goiás state, Brazil. Initially, RNA samples were obtained from swab samples from 15 patients from the state of Goiás, Brazil, in November/2020 and February/2021 to validate protocol steps. The libraries were prepared following AmpliSeq for Illumina workflow with modifications; subsequently, we analyzed 305 positive samples collected from the state of Goiás from December 2020 to July 2021. For protocol improvement, we removed the need to treat samples with DNAse and demonstrated the importance of quantification by qPCR before and after library dilution. No fragmentation pattern was observed in the samples analyzed with Bioanalyzer. The libraries returned sequencing results that were used for genome assembly and variant detection. We were able to assemble SARS-CoV-2 genomes from 318 samples, which were used to identify 13 variants of coronavirus circulating in Goiás throughout those months. Variants of concern, such as Alpha (B.1.1.7), Gamma (P.1) and Delta (B.1.617.2) were detected; the latter was detected at first in Goiás in April 2021. The modifications in the workflow we developed were successfully applied to detect SARS-CoV-2 variants, resulting in high coverage genome assembly, and they can be used to increase the number of genome sequences and aid in epidemiological surveillance in Brazil.

Genet. Mol. Res. 21(1): GMR19018
DOI: 10.4238/gmr19018
Research Article

Nitrogen management distinctly influences the expression and magnitude of relationships between wheat ear components and grain yield. We sought to determine the variables linked to wheat ear that are more responsive to changes in the dose and form of nitrogen supply and to determine the direct and indirect effects on grain yield by single and fractional nitrogen supply in soybean/wheat and corn/wheat succession systems. The study was conducted in 2018 and 2019 in a randomized block experimental design with four replications, in a 3 x 3 factorial, for N-fertilizer doses (30, 60, 120 kg ha-1) and forms of supply [single dose (100 %) in phenological stage V3 (third expanded leaf); fractionated dose (70% and 30%) at the V3/V6 phenological stage (third and sixth expanded leaf) and; fractionated dose (70% and 30%) at phenological stage V3/R1 (expanded third leaf and beginning of grain filling)], respectively, in the soybean/wheat and corn/wheat succession systems. The increase in the nitrogen doses promotes productivity due to the greater contribution of grain mass and ear length in the V3 stage and ear grain mass in V3/V6 and V3/R1, in a soybean/wheat system. In the corn/wheat system, the increase in nitrogen promotes productivity, with a greater contribution of grain mass in the ear, regardless of the form of supply. Nitrogen supply in single dose (V3) and fractionated in theV3/V6 gave similar productivity, with a reduction in V3/R1. The grain mass of the ear showed greater contribution of alteration by the form of single and fractioned nitrogen supply, regardless of the dose and succession system. The grain mass of the ear shows a high correlation with yield, regardless of the dose and form of nitrogen supply in the soybean/wheat system, with a positive indirect effect by the ear mass. In the corn/wheat system, ear grain mass shows a high correlation with yield, when nitrogen is supplied in a single dose at 30 and 60 kg ha-1, with a positive indirect effect due to ear length.

Genet. Mol. Res. 21(1): GMR19008
DOI: 10.4238/gmr19008
Research Article

Glutathione S‐transferases are detoxification enzymes that protect cells from oxidative stress and help maintain genomic integrity. GSTM and GSTT family genes may be deleted, causing reduced or no glutathione S-transferase activity so that electrophilic carcinogens cannot be eliminated efficiently. This genetic alteration affects cancer incidence and prognosis. We genotyped GSTM1 and GSTT1 in 87 Brazilian leukemia patients by multiplex PCR, divided into acute or chronic, and lymphocytic or myeloid type cancers. GSTM1-null and GSTT1-null frequency was significantly higher in chronicmyeloid leukemia (67.65% and 46.20%) and chroniclymphoid leukemia (29.41% and 12.30%) in relation to controls, respectively, than in non-leukemia controls. More than that, double null genotyping was significantly more present in acutelymphoid leukemia than controls. When individual GSTM1 and GSTT1 genotyping were analyzed, again CML and CLL presented significantly diferent in genotyping frequency compared to controls (11.11% and 3.8%). Double null genotypes were significantly more frequent in acute lymphoid leukemia than in controls. Furthermore, CML patients presented a statistically significant higher percentage of double-null and GSTM1-null genotypes, and CLL patients had a significantly higher frequency of GSTT1-null genotyping when compared to controls. We suggest that GST genotypes are an important risk factor for leukemia development in the Brazilian population, especially in chronic leukemia due to inefficient detoxification of oxidative stress products.

Genet. Mol. Res. 21(1): GMR18994
DOI: 10.4238/gmr18994
Research Article

Cardiac fibrosis is common and detrimental in numerous heart diseases, affecting millions of patients worldwide. Cardiac fibrosis is characterized by excessive production of extracellular matrix (ECM) constituents such as fibrillar collagens, produced by activated cardiac fibroblasts, i.e.: myofibroblasts. Therapeutic targeting of cardiac fibrosis is highly attractive; however, it remains a major medical challenge. Fibromodulin is a small leucine-rich proteoglycan localized in the ECM. Fibromodulin binds to collagen fibrils, and plays a critical role in collagen fibrillogenesis, ECM organization, wound healing and regulation of the pro-fibrotic cytokine transforming growth factor beta (TGFβ) in several organs. Fibromodulin is highly upregulated in mice and patients with heart failure, but little is known about its role in cardiac fibrosis. Our recent findings from primary cultures of cardiac fibroblasts from neonatal rat hearts suggest that fibromodulin has anti-fibrotic effects. Here we investigated the translational value of these findings by overexpressing fibromodulin in cultured human fetal and adult cardiac fibroblasts. The effects of fibromodulin overexpression on gene expression were measured by qPCR and gene arrays, whereas protein levels were measured by Western blotting, and collagen synthesis by radioactive proline incorporation. The results support our previous findings and indicate relevance for human disease. We found that fibromodulin reduced the expression levels of the collagen cross-linking enzymes lysyl oxidase (LOX) and transglutaminase 2 (TGM2). Fibromodulin also reduced the levels of connective tissue growth factor (CTGF) and periostin (POSTN), indicating reduced TGFβ activity. Reduced levels of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) suggested reduced potential for immune cell adhesion, and gene arrays indicated altered integrin expression, suggesting altered ECM-cell adhesion. Expression of fibrillar collagens was unaffected. In conclusion, fibromodulin reduced TGFβ activity and down-regulated central collagen-crosslinking enzymes, in line with an anti-fibrotic effect of fibromodulin in human cardiac fibroblasts.

Genet. Mol. Res. 21(1): GMR19007
DOI: 10.4238/gmr19007

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