Research Article

Table of Contents | Genet. Mol. Res. 2019 (2)

Displaying 1 - 15 of 45
Research Article

The common bean is widely distributed throughout Brazil and within Mato Grosso state. It has wide genetic variability and adaptability to various environmental conditions. We evaluated the genetic divergence and agronomic performance of common bean genotypes in order to initiate a bean breeding program in the region of Cáceres, MT. We included 58 genotypes (40 traditional accessions, 14 lines and four cultivars) using a randomized block design with three replications, at Empresa de Pesquisa, Assistência e Extensão Rural do Mato Grosso, evaluating 10 morphological and agronomic characteristics. The data were submitted to analysis of variance and cluster analysis based on Mahalanobis distance; we used the Tocher and UPGMA grouping methods to group to the genotypes. Through analysis of variance, we detected significant differences at 1% probability for all traits. The shortest genetic distance was found between genotypes 13 and 26 (= 1.51) as the most similar, while 20 and 4 ( = 346.00) were the most dissimilar. The Tocher and UPGMA clustering methods were similar in grouping the genotypes, with the most divergence in different groups. Crossing of the earliest genotypes (11, 13 and 16) with the improved genotypes (43 and 51) resulted in the greatest heterotic effect, increasing the likelihood of the emergence of superior genotypes in the progenies, which is beneficial for the initiation of the bean breeding program for the Cerrado region of Cáceres in Mato Grosso.

 

Genet. Mol. Res. 18(2): GMR18299
DOI: 10.4238/gmr18299
Research Article

Serum paraoxonase (PON1) and hemochromatosis (HFE) genes may play important roles in lead toxicity owing to their role in xenobiotic and iron metabolism, respectively. The association between PON1 and HFE genotypes and blood lead levels (BLLs) was examined in lead exposed subjects from Saudi Arabia. The polymorphisms at PON1 L55M, PON1 Q192R, HFE H63D and HFE C282Y (using PCR-RFLP) and their relation to BLLs was evaluated. The recruited subjects (N = 127) were categorized into low BLL group (<10 μg/dL) and high BLL group (>10 μg/dL) according to CDC guidelines. The low BLL group had a mean level of 3.94 µg/dL, while the high BLL group had levels of 15.33 µg/dL (P < 0.001). Overall, the genetic variants, TA and AA in the PON1 L55M were significantly (P = 0.00002 and P = 0.00322, respectively) associated with a risk of lead toxicity and the allele ‘A’ was a risk factor (P = 0.00002). However, the Q192R genotype of PON1 along with HFE H63D and HFE C282Y did not significantly increase the risk of developing lead poisoning. Our findings suggest that L55M gene polymorphism influences the susceptibility of subjects to lead exposure and thus it could be a useful biomarker of genetic susceptibility in assessing an individual’s risk of damage from heavy metal exposure.

 

Genet. Mol. Res. 18(2): GMR18317
DOI: 10.4238/gmr18317
Research Article

Several strategies have been employed in the breeding of passion fruit with a view to the generation of superior progeny. In an effort to develop more precise methods in breeding, we compared the efficiency of the Post-Hoc Blocking Row-Col technique, which is an a posteriori technique that consists of the overlapping of a block structure on the original-field design, with a randomized-block design and compared different selection strategies within and among half-sib families, using the REML/BLUP mixed-model methodology. Twenty-three half-sib families from the third cycle of recurrent selection of the breeding program of Universidade Estadual do Norte Fluminense Darcy Ribeiro - UENF were evaluated. The trial took place in the experimental unit of UENF, in Itaocara - RJ, Brazil. Plants were trained on vertical stakes, with four replicates and three plants per plot. They were assessed individually for the traits number of fruits per plant, fruit mass per plant, fruit length, fruit diameter, peel thickness, total soluble solids, pH, pulp percentage, and production per plant. No significant difference was found in the test of efficiency of the designs for any of the evaluated traits. Within-family heritability (h2ad) had a similar magnitude to individual heritability (h2a), indicating that even in the 4th cycle of recurrent selection, genetic variability still exists within the evaluated progeny. Selection within half-sib families provided superior gains when compared with selection among families for the traits number of fruits; production; fruit mass, length, and diameter; total soluble solids; pH; and pulp percentage. The best selection strategy was within families, as it generated higher selection-gain estimates than those obtained with selection between families and the direct-selection and index-selection approach.

Genet. Mol. Res. 18(2): GMR18305
DOI: 10.4238/gmr18305
Research Article

The development of common bean cultivars with upright plant architecture and high grain yield meets the needs of bean growers by facilitating culture management and mechanized harvest. The objectives of this study were to evaluate whether Mesoamerican common bean lines differ for upright plant architecture traits and grain yield, analyze correlations between these traits, and to select superior common bean lines based on a selection index. Four experiments were carried out in different years and growing seasons at low altitude (95 m above sea level) in southern Brazil. These experiments were composed of 17 carioca and black bean inbred lines, which are representative of the most widely produced and consumed common bean classes in Brazil. Upright plant architecture was assessed by 12 characters, and the resulting data were subjected to individual and joint analyses of variance, correlation, and selection index (rank sum). A significant effect for genotype and/or genotype x environment interaction was observed for all traits, except for the length of the first internode and hypocotyl diameter. Most plant architecture traits were correlated, indicating that it is possible to reduce the number of traits that need to be evaluated. Grain yield was negatively correlated with the lengths of the fourth (r = -0.574) and fifth internodes (r = -0.641). General adaptation score, insertion of the first pod, and lengths of the fourth and fifth internodes were efficient to characterize upright plant architecture in the common bean. Indirect selection by the shortest length of the fourth and fifth internodes was efficient to increase grain yield. The genotypes LP 11-117, SM 0312, BRS Valente, and Guapo Brilhante were selected based on a rank sum index. These genotypes present upright plant architecture and high grain yield, which is a common bean ideotype suitable for cultivation at low altitude in southern Brazil.

 

Genet. Mol. Res. 18(2): GMR18323
DOI: 10.4238/gmr18323
Research Article

VIRB proteins from Brucella spp. constitute the type IV Secretion System (T4SS), a key virulence factor that mediates the intracellular survival of these bacteria. We investigated the immunogenicity and protection of proteins produced by the virB9 and virB12 genes in the DNA vaccine and DNA prime-protein boost strategies. Groups of 10 mice were vaccinated with pcDNAvirB9, pcDNAvirB12, pcDNAvirB9+rVIRB9 or pcDNAvirB12+rVIRB12. The latter two groups were vaccinated with the proteins rVIRB9 and rVIRB12, respectively, during the third immunization. Three weeks after the last immunization, six animals from each group were challenged intraperitoneally with B. abortus strain S2308, and the efficacy of the vaccines was calculated as the log10 of protection by subtracting the mean log CFU of the vaccinated group from the mean log CFU of the negative control group (injected with sterile saline). Most of the vaccinated mice produced total IgG and the subclasses IgG1 and IgG2a against the respective protein, except for the mice vaccinated with pcDNAvirB12. Cytokines IFN-γ and IL-10 were produced, but without a significant difference between the vaccinated and negative control groups. The vaccines did not induce significant levels of protection, in contrast to the immunization obtained with the S19 vaccine strain (Log10, 1.48). In conclusion, the virB9 and virB12 genes of B. abortus, using DNA vaccine and DNA prime-protein boost strategies, were able to induce both humoral and cellular immune responses, but not enough to induce significant protection in the immunized mice. However, given the response in this system, further investigations using the virB9 and virB12 genes of Brucella spp., together with different immune modulators, are warranted. An effort should be made to direct and enhance the immune response, in order to identify a combination that stimulates a better immune response and, consequently, a better level of protection.

Genet. Mol. Res. 18(2): GMR18295
DOI: 10.4238/gmr18295
Research Article

Xanthomonas albilineans causes leaf scald disease, one of the most economically important diseases of sugarcane. Leaf scald is a systemic and vascular disease that can severely reduce productivity. The best and primary method of leaf scald control is the use of tolerant cultivars. Knowing that cell wall lignification is a plant defense mechanism and that chorismate mutase is relevant in this process, we analyzed the expression pattern of the chorismate mutase 2 (SoCM2) gene in two contrasting sugarcane cultivars by RT-qPCR.  A similar pattern of downregulation for the SoCM2 gene was observed in both cultivars; however, after 72h, the tolerant cultivar (RB867515) showed repression of the SoCM2 gene, while for the susceptible cultivar (SP78-4467), SoCM2 gene downregulation occurred only after 144h. Here we propose that early repression of the SoCM2 gene is responsible for tolerance to X. albilineans infection in the sugarcane cultivar RB867515.

 

Genet. Mol. Res. 18(2): GMR18262
DOI: 10.4238/gmr183262
Research Article

Myelodysplastic syndrome (MDS), an onco-hematological disease, is characterized by distinct levels of peripheral blood cytopenia, cell differentiation dysplasia and various types of chromosomal alterations. The Revised International Prognostic Scoring System uses the GTG-banding karyotype as one of the main components for scoring patient prognosis in MDS. Using GTG-banding karyotyping, we looked for chromosomal alterations in bone marrow samples obtained from a cohort of Brazilian patients in Goiás state, Brazil. Numerical and/or structural chromosomal alterations were detected in 15 of 29 patients. A total of 23 clones with the chromosomal alterations were obtained, of which 12 were numerical and 11 were structural. Complete trisomies were observed in five clones, complete monosomies in three clones, triploidies in three clones, and one clone contained a marker chromosome. Among the clones with structural alterations, two clones had a partial trisomy in 1q, five clones had partial monosomies, one clone contained an isochromosome, and three clones showed reciprocal translocations. The high diversity of chromosomal alterations is inherent to the strong degree of chromosomal and genomic instability in this myeloid disease. These alterations can be associated with the activation or inhibition of gene expression, leading to the deregulation of critical cell viability functions and hence ineffectiveness of hematopoiesis in MDS. Our study is the first to identify chromosomal alterations associated with this poorly studied disease in central Brazil.

Genet. Mol. Res. 18(2): GMR18322
DOI: 10.4238/gmr18322
Research Article

The etiopathology of male infertility is highly complex, involving gene - environment interactions to regulate spermatogenesis. Consequently, genetic analysis becomes imperative for cases of non-obstructive azoospermia (NOA) to identify the causative factors. Cases (n = 111) of NOA referred to the cytogenetics and molecular genetics laboratory of the All India Institute of Medical Sciences in -Patna from 2013-2018 were subjected to 1) karyotyping using GTG bandings techniques, 2) fluorescence in situ hybridization (FISH) for the sex determining region (SRY), and 3) PCR based analysis of STS markers based on microdeletion of the Y- chromosome after isolation of genomic DNA from whole blood. A flow cytometer was used for a cell- kinetic and DNA methylation study after incorporation of 5-azacytidine (5-AzaC) (1.0 ug/mL) in lymphocyte culture. PCR products were analyzed on an agarose gel (1.5%) and bands were visualized on Gel Doc after ethidium bromide staining. Chromosomal abnormalities, including structural numerical variations, were observed in 14 of the karyotypes. Eight cases showed a 46,XY/47,XYY i.e. mosaic pattern; two cases 46, XY/45/XO; a single case with 47,XY +16; two cases with 46,X+ ring Y; a single case with 46,XY+dicentric in chromosome-7; and two cases showed a normal 46,XY karyotypic pattern. Cellular proliferation increased after incorporation of 5-azaC (1.0 µg/mL) for 24 h in lymphocyte culture at the S - phase of the cell cycle. Out of 14 cases analyzed with FISH, three cases showed loss of the SRY region, amongst which two cases were SRY negative (-ve) with a normal 46, XY karyotype. Serum follicle stimulating hormone values were higher (21 U/L) in cases of mosaicism as compared to normal individuals. The frequency of microdeletion deletion of the AZFc region varied in different cases; one case of infertility showing deletion of the Sy 267 STS marker (102bp); loss of a 350bp band (Sy 254) was found in all the cases of infertility. These genetic variations are responsible for dysregulation of spermatogenesis leading to infertility in males. Genetic counselling would be relevant in such cases, before beginning assisted reproductive techniques such as in vitro fertilization.

 

Genet. Mol. Res. 18(2): GMR18349
DOI: 10.4238/gmr18349
Research Article

Mannose-binding lectin (MBL) is a lectin complement protein encoded by the MBL2 gene that has an important role in the control of infections caused by intracellular pathogens. However, there is no consensus about the effect of MBL2 polymorphism and MBL levels in leishmaniasis infections. We investigated the implications of MBL2 gene variants as well as MBL serum levels and occurrence of visceral leishmaniosis (VL) caused by Leishmania chagasi. A case-control analytic study was performed on 161 patients with VL and 161 healthy individuals in a northeast region of Brazil. The alleles of exon 1 (MBL2*A, MBL2*B, MBL2*C and MBL2*D) and promoter region (-550L/H, -221Y/X and +4P/Q) were identified by automatic sequencing and the MBL serum levels were determined using an ELISA kit. MBL serum levels were similar in VL patients compared to the healthy controls. Also, allelic, genotype and haplotype frequencies of variants in exon 1 and promoter region did not significantly differ between case and control groups. Overall, our data show that MBL2 polymorphisms within the structural gene as well as the promoter region influence functional MBL serum levels but are not associated with susceptibility to L. chagasi infection in this population. We observed a lack of consensus regarding the association of MBL 2 polymorphisms with leishmaniasis worldwide, indicating that the influence of genetic variations can differ in different populations and with differences in parasite/vector relationship.

Genet. Mol. Res. 18(2): GMR18148
DOI: 10.4238/gmr18148
Research Article

The Allium genus stands out for its uses in human food and also for its medicinal properties. Many representatives of the Amaryllidaceae family are known for producing mannose binding lectins (MBL). In plants, lectins act as reserves of proteins that can be used for plant growth and development and also in defense against herbivores and pathogens, being toxic to some aphids and sucking insects. We examined physicochemical characteristics, such as isoelectric points and hydropathicity, of 22 sequences of MBL protein from Allium species and from other representatives of the Amaryllidaceae family present in public databases. Phylogenetic analysis, identification of functional domains and 3D homology modeling were also performed. We found two conserved functional motifs in the MBL sequences. It was observed that for all species the MBL had a hydrophilic character and great variation in isoelectric points. The phylogenetic analysis was not consistent with the taxonomic classification of the species evaluated at the infrageneric level. However, the methods proved efficient for the separation up to the level of tribes within the Amaryllidaceae family. The generated 3D models also provide a better understanding of their tertiary structures and molecular functions.

Genet. Mol. Res. 18(2): GMR18187
DOI: 10.4238/gmr18187
Research Article

With the advent of the Brazilian Government Biodiesel Program and the increasing demand for vegetable oils, Jatropha curcas has been emphasized as an alternative raw material. Given the known water stress tolerance of this species, we evaluated the plasticity of photosynthetic metabolism in nine genotypes of J. curcas under water stress (WS). The hypotheses that (1) the photosynthetic metabolism of J. curcas is plastic and changes towards crassulacean acid metabolism  (CAM)  according to water availability and (2) the plasticity of the metabolic alteration is variable among the different genotypes and is related to physiological adjustments, were tested. Water deficit led to 167 and 187% increase in phosphoenolpyruvate carboxylase (PEPC) activity in leaves collected at 4 and 16 h,, respectively, when compared to control plants. The accumulation of organic acids (malate and citrate) differed among genotypes and WS treatments. There was an average increase of 270% for plants submitted to WS, when citrate was compared to malate. There was an increase of 62% in total soluble sugar content and a decrease of 27% in starch content in plants under WS when compared to controls. Significant effects of genotype and watering regime were detected for carbon isotope composition of leaf biomass. We detected changes in photosynthetic metabolism towards low CAM levels, thus explaining the maintenance of efficiency of water use under water deficit.

 

Genet. Mol. Res. 18(2): GMR18228
DOI: 10.4238/gmr18228
Research Article

In passion-fruit breeding programs, characterizing genotypes developed through morpho-agronomic descriptors helps quantify genetic diversity and identify individuals with desirable qualities. We examined the discriminatory ability of passion-fruit descriptors and determined their relative importance in the characterization of 91 genotypes from a breeding program for resistance to Cowpea aphid-borne mosaic virus. Twenty-four quantitative and 14 multi-category qualitative descriptors related to plant, leaf, flower, and fruit characteristics were used. The quantitative descriptors were subjected to correlation and principal-component analyses and selected based on direct selection and the Singh method. The traits were used to obtain a distance matrix, based on Gower’s algorithm, and a comparative clustering between the dendrograms for the morpho-agronomic variables was obtained using the unweighted pair group method with arithmetic mean procedure. Based on the principal components method, the traits that most contributed to genetic variability were number of seeds (23.241%), petiole length (19.438%), and petal width (10.440%). In its turn by the Singh method, the traits androgynophore length (6.68%), followed by flower length (5.47%), area under the disease progress curve (5.27%), and peduncle length (5.17%) were those which most contributed to the differentiation of genotypes. Although seven descriptors (leaf length, bract width, corona long filament length, sepal length, fruit width, fruit mass, and mass of fruit pulp) showed little contribution to the characterization of genotypes, their discard is not suggested, as they significantly contribute to the discrimination of genetic divergence in the population. Comparative analysis between the dendrogram containing all descriptors and the dendrogram containing only flower, leaf, or fruit descriptors evidenced the need for using a large number of descriptors in the characterization of genetic diversity in Passiflora. The use of all 38 descriptors increased the efficiency in the discrimination of groups.

Genet. Mol. Res. 18(2): GMR18255
DOI: 10.4238/gmr18255
Research Article

The black rhino sanctuaries system has played a key role in repopulating and starting new subpopulations in Kenya. If this system is efficiently managed it may save the black rhinoceros from local extinction. Understanding the genetic status of endangered species is the most elemental sine qua non of animal breeding and conservation. It is therefore important to determine the genetic diversity of black rhino populations, especially of nucleus breeding populations that are used as a source of individuals for translocation and supplementation programs. We assessed the genetic diversity of one of the pioneer breeding subpopulations of the black rhino Diceros bicornis in Kenya using a mitochondrial DNA D-loop region. We then compared this subpopulation with the entire Kenyan population to determine its status vis-à-vis the Kenya pooled population and determine the possible sources/relationships of the founder individuals. In the 469-bp D-loop region we analyzed, 7.11% of the sites were variants, contributing to 18 distinct haplotypes. Estimates of genetic diversity using haplotype and nucleotide diversity metrics showed that the Lake Nakuru National Park subpopulation has a slightly lower genetic diversity when compared with that of the pooled Kenya population.  The phylogenetic tree revealed that Lake Nakuru National Park founder individuals were probably sourced from multiple subpopulations. The dendrogram and the principal coordinate analysis plot indicated that the Maasai Mara subpopulation is not a distinct subpopulation, as had been suggested previously. Our results provide baseline genetic data for the Lake Nakuru National Park breeding subpopulation and valuable information for translocation/supplementation programs.

Genet. Mol. Res. 18(2): GMR18203
DOI: 10.4238/gmr18203
Research Article

Human leukocyte antigen (HLA) G, a non-classical HLA class I (MHC class I) molecule, is characterized by a low degree of polymorphism, unlike classical MHC-I. The 14-base pair insertion/deletion (indel) polymorphism (rs16375) in exon 8 of the 3′ untranslated region has been reported to control HLA-G expression transcriptionally and post-transcriptionally. We evaluated a possible association between the 14-bp indel polymorphism of HLA-G and the level of this protein in serum [sHLA-G1] and in primary-tumor tissue in colorectal cancer (CRC) of a Saudi population. A total of 105 patients with CRC and 105 healthy controls were analyzed for the 14-bp indel polymorphism. sHLA-G1; histological presence of HLA-G was also investigated for association with CRC. Lower prevalence of the heterozygous genotype of the 14-bp indel polymorphism was observed among the patients with CRC, though the difference was not quite significant (P = 0.052). In addition, the sHLA-G1 pattern did not show a significant difference between the patients with CRC and the controls. However, the expression pattern of HLA-G in colorectal tissue showed a heterogeneous profile, marked by a lack of expression in colorectal adenocarcinoma and normal cells and focal expression of the protein in the transition zone.

 

Genet. Mol. Res. 18(2): GMR18324
DOI: 10.4238/gmr18324
Research Article

Glioblastomas are the most common primary malignant brain tumors, and despite current advances in therapy, they are still extremely resistant to treatment. The tumorigenic potential of glioblastomas is due to a subpopulation of neoplastic cells, called cancer stem cells (CSCs), which in culture form a suspension cluster called neurospheres. Their properties include tumorigenesis, chemoresistance, and radioresistance. Several miRNAs exhibit altered expression levels during gliomagenesis; thus they constitute one of the key mechanisms for CSC regulation. We evaluated whether temozolomide and ionizing radiation modulate the expression of the tumor suppressor microRNAs-15, -16 and of the oncogenic miR-21 in neurospheres and attached cells in a glioblastoma cell line culture (U343-MG). The methods included staining with Trypan blue to verify cell viability and real time PCR to quantify the expression of microRNAs after exposure to  treatments. We observed that miR-16 was more highly expressed in neurospheres than in attached cells 48h after treatment with temozolomide, and miR-21 was highly expressed in attached cells compared to neurospheres treated with temozolomide at time 0 h (30 min after treatment); at 48 h this microRNA was highly expressed in neurospheres treated with temozolomide in association with ionizing radiation. We concluded that the microRNAs were differentially expressed when comparing the different cell types and treatment modalities. The higher expression of miR-21 in neurospheres at time 48 h may suggest a pathway of CSC radioresistance and chemoresistance, with further rapid tumor growth and recurrence or resistance to other treatment modalities.

 

Genet. Mol. Res. 18(2): GMR18095
DOI: 10.4238/gmr18095

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