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Research Article

We used next-generation sequencing technology to characterize 19 genomic simple sequence repeat (SSR) markers and 11 expressed sequence tag (EST) SSR markers from Leuciscus leuciscus baicalensis, a small freshwater fish that is widely distributed in Xinjiang, China. Primers were used to test for polymorphisms in three L. leuciscus baicalensis populations in Xinjiang. There were 4-27 (average 11.3) alleles (N), the expected heterozygosity (H) was 0.36-0.94 (average 0.75 ± 0.14), the observed heterozygosity (H) was 0.37-1.00 (average 0.68 ± 0.18), and the polymorphism information content (PIC) was 0.31-0.93 (average 0.71). The averages of H and PIC for the EST-SSR markers were slightly lower than for the genomic SSR markers. Genetic analysis of the three populations showed similar results for PIC, H, and N. Amplifications were performed in nine other species; the top three transferability values were for Rutilus lacustris (80%), Leuciscus idus (76.7%), and Phoxinus ujmonensis (63.3%), with the following average values: PIC (0.56, 4.46, and 0.52); N (0.40, 3.00, and 0.32); and H (0.44, 2.74, and 0.22), respectively. L. leuciscus baicalensis is one of the most important commercial fish in Xinjiang, but in recent years, fishery resources have decreased sharply owing to water conservation projects, unreasonable utilization, and invasion by alien species. These novel SSR markers are appropriate for studies involving fingerprinting, gene flow, genetic diversity, population structure, and molecular-assisted breeding, and could contribute to the conservation of L. leuciscus baicalensis.

Genet. Mol. Res. 2017
DOI: 10.4238/gmr16029376
Research Article

Atherosclerotic and its cardiovascular complications are responsible for 17.5 million deaths a year, according to the World Health Organization. There is consensus that atherosclerosis involves multiple pathogenic processes initiated by endothelial dysfunction, with inflammation and vascular proliferation determining alterations in the matrix, with consequent formation of the atheromatous plaque and its clinical implications. Risk factors such as hypertension, diabetes mellitus, dyslipidemia, and smoking are widely known. Currently, genotyping, which is not directly related to these factors, is not accepted to estimate the risk of cardiovascular diseases, but strong evidence indicates several polymorphic genes as factors of risk and progression leading to complications of the disease. Among the genes involved, eNOS (endothelial nitric oxide synthase gene), which is responsible for the production of endothelial nitric oxide (an important arterial vasodilator), when presented in polymorphic variation can determine production, malfunction, and predisposition to atherosclerosis. In the present study, we analyzed the G894T polymorphism of the eNOS gene in groups of individuals diagnosed with atherosclerosis and in a control group. We collected 200 blood samples from patients previously diagnosed with atherosclerosis and 100 samples formed the control group. The genotyping analysis for polymorphism of the eNOS gene was determined by PCR. We considered variables such as gender, smoking, smoking history, and alcohol consumption; statistical differences were found in the distribution of case and control groups (P = 0.0378) and in non-smoking patients (P = 0.0263). In the other associations, no statistically significant difference was found. In the population studied, the frequency of the heterozygous genotype (GT) was much higher than in the other populations (GG and TT) in both groups (case and control). The GG genotype showed greater susceptibility to atherosclerosis. Association of the GG genotype in non-smokers also showed greater susceptibility. Gender, alcohol consumption, smoking, and smoking history did not influence atherosclerosis.

Genet. Mol. Res. 2017
DOI: 10.4238/gmr16029550
Research Article

The goal of this study was to analyze the genetic divergence among Psidium species accessions based on SNPs developed for Eucalyptus. Fifty-three Psidium accessions, including 47 P. guajava, were genotyped with EUCHIP60K. The dendrogram similarity ranged from 0.58 to 1.00, with a cophenetic value of 0.97. Five groups were identified at dendrogram cut point of 0.7: the first with 44 guava accessions, the second with 1 guava accession, the third with 3 P. guineense accessions, the forth with 2 guava accessions, and the fifth with 3 P. cattleianum accessions. The Bayesian analyses suggested seven subpopulations, with formation of two additional groups with guava accessions. Primers designed with Eucalyptus SNP sequences resulted in reliable Psidium amplicons on 6% polyacrylamide gels. In general, the SNP dendrogram agreed with biological genus structure, since different species were not grouped, indicating that transferability among Myrtaceae genus was possible and reliable.

Genet. Mol. Res. 2017
DOI: 10.4238/gmr16029566
Research Article

Micropropagation is an important tool for large-scale multiplication of plant superior genotypes. However, somaclonal variation is one of the drawbacks of this process. Changes in DNA methylation have been widely reported as one of the main causes of somaclonal variations in plants. In order to investigate the occurrence of changes in the methylation pattern of sugarcane somaclonal variants, the MSAP (methylation-sensitive amplified polymorphism) technique was applied to micro-propagated plantlets sampled at the third subculture phase. The mother plant, in vitro normal plantlets, and in vitro abnormal plantlets (somaclonal variants) of four sugarcane clones were screened against 16 MSAP selective primers for EcoRI/MspI and EcoRI/HpaII restriction enzymes. A total of 1005 and 1200 MSAP-derived markers with polymorphism percentages of 28.36 and 40.67 were obtained for EcoRI/HpaII and EcoRI/MspI restriction enzyme combinations, respectively. The genetic similarity between the mother plant and the somaclonal variants ranged from 0.877 to 0.911 (EcoRI/MspI) and from 0.928 to 0.955 (EcoRI/HpaII). Most of the MASPs among mother plant and micro-propagated plantlets were derived from EcoRI/MspI restriction enzymes suggesting alteration due to gain or loss of internal cytosine methylation. A higher rate of loss of methylation (hypomethylation) than gain of methylation (hypermethylation) was observed in the abnormal in vitro sugarcane plantlets. Although changes in the methylation pattern were also observed in the in vitro normal plantlets, they were lower than those observed for the in vitro abnormal plantlets. The MASP technique proved to be a promising tool to early assessment of genetic fidelity of micro-propagated sugarcane plants.

Genet. Mol. Res. 2017
DOI: 10.4238/gmr16029585
Research Article

Given the importance of selecting lines to obtain hybrids, we aimed to verify the relationship between morphological traits that can be used as the criteria for the selection of sorghum lines with high grain yield and earliness. A total of 18 traits were evaluated in 160 sorghum elite lines, in an incomplete block design with two replicates. A correlation network was used to graphically express the estimates of phenotypic and genotypic correlations between the traits. Two path analyses were processed, the first considering grain yield and the second considering flowering as the principle dependent variable. In general, most of the variation in the grain yield and flowering of sorghum lines was explained by the traits evaluated. Selecting sorghum lines with greater width of the third leaf blade from flag leaf, panicle weight, and panicle harvest index might lead to increased grain yield, and selecting sorghum genotypes with higher plant height might lead to reduced earliness and increased grain yield. Thus, the results suggest the establishment of selection indices aiming at simultaneously increasing the grain yield and earliness in sorghum genotypes.

Genet. Mol. Res. 2017
DOI: 10.4238/gmr16029649
Research Article

Cariniana estrellensis is one of the largest trees found in Brazilian tropical forests. The species is typical of advanced stages of succession, characteristic of climax forests, and essential in genetic conservation and environmental restoration plans. In this study, we assessed Mendelian inheritance, genetic linkage, and genotypic disequilibrium in nine microsatellite loci for a C. estrellensis population. We sampled and genotyped 285 adult trees and collected seeds from 20 trees in a fragmented forest landscape in Brazil. Based on maternal genotypes and their seeds, we found no deviation from the expected 1:1 Mendelian segregation and no genetic linkage between pairwise loci. However, for adults, genotypic disequilibrium was detected for four pairs of loci, suggesting that this result was not caused by genetic linkage. Based on these results, we analyzed microsatellite loci that are suitable for use in population genetic studies assessing genetic diversity, mating system, and gene flow in C. estrellensis populations.

Genet. Mol. Res. 2017
DOI: 10.4238/gmr16029653
Research Article

Single nucleotide polymorphisms (SNPs) are the best genetic markers for associative studies of the immune system in invertebrates. In the marine shrimp Litopenaeus vannamei, SNPs linked to disease resistance have been reported for some genes, such as hemocyanin, anti-lipopolysaccharide factor, and heat-shock protein 70 (Hsp70). In the present study, polymorphisms in the Hsp70 gene were investigated among three commercial L. vannamei populations bred in Northeast and South Brazil. The first population withstood a strong white spot disease outbreak; the second population suffered extended exposure to infectious myonecrosis; the third population was a high health population, which was experimentally infected with white spot syndrome virus (WSSV) in the present study. All five previously known SNPs (C661A, T712C, C782T, C892T, and C1090T) were detected in the coding region of Hsp70, by Sanger sequencing of 119 shrimp. Significant differences in genetic and genotype frequencies among populations were observed for C661A, C892T, and C1090T. In the population submitted to WSSV challenge, no frequency differences were found between dead and surviving shrimp groups. These results indicate that the Hsp70 polymorphisms described here cannot be associated with WSSV tolerance. However, significant frequency differences were observed for the population exposed to infectious myonecrosis virus. This is the first time that L. vannamei Hsp70 gene polymorphisms were studied in correlation with these important shrimp viruses.

Genet. Mol. Res. 2017
DOI: 10.4238/gmr16029668
Research Article

We aimed to analyze gastric signet ring cell (SRC) carcinoma subtypes by investigating gastric and intestinal phenotypic marker expression, and explore the relationship between phenotype and K-ras mutation. Immunohistochemistry was performed on 163 SRC carcinoma patient specimens to detect gastric (MUC1, MUC5AC, and MUC6) and intestinal (MUC2 and CDX2) phenotypic markers, and tumors were classified into gastric (G), intestinal (I), and gastrointestinal (GI) phenotypes. DNA was extracted from the formalin-fixed, paraffin-embedded tumor samples, and K-ras mutations in codons 12, 13, and 61 were identified using polymerase chain reaction-based direct DNA sequencing. G, GI, and I phenotypes were observed in 63 (38.6%), 71 (43.5%), and 29 cases (17.8%), respectively. Expression of MUC2 was significantly associated with invasion depth and lymph node metastasis (P = 0.001 and 0.002, respectively), whereas that of CDX2 significantly corresponded to tumor size and submucosal invasion (P = 0.004 and 0.001, respectively). MUC5AC expression was inversely associated with gastric wall invasion (P = 0.001). Intestinal phenotypic marker expression was positively associated with gastric wall invasion and lymph node metastasis. K-ras mutations, all of which were in codon 12, were detected in 20 (12.27%) tumors, were significantly associated with the I phenotype, and exhibited an inverse relationship with MUC5AC and MUC6 expression. I-phenotype SRC carcinomas should be distinguished from those of the G phenotype because of their increased malignancy regarding invasion and metastasis, and higher K-ras aberration rate. The different K-ras mutation frequencies observed imply distinct genetic mechanisms in the carcinogenesis of I- and G-phenotype gastric SRC carcinomas.

Genet. Mol. Res. 2017
DOI: 10.4238/gmr16029181
Research Article

Childhood obesity is a serious public health concern condition, as excess body fat can negatively affect a child's health. Obestatin is a hormone that regulates body weight by suppressing appetite and reducing food intake; fasting obestatin level is negatively correlated with basal insulin level. This study aims to investigate the role of obestatin in insulin resistance. A total of 54 children with simple obesity and 57 healthy controls were recruited. Levels of serum insulin, fasting blood glucose, cholesterol, triglyceride, low-density lipoprotein, and high-density lipoprotein were measured. Serum obestatin level was determined using an enzyme-linked immunosorbent assay. We found that body mass index and waist-hip ratio of obese children were significantly higher, while obestatin level in the obese group was significantly lower (P < 0.001), as compared to those of controls. In addition, obese children exhibited higher levels of insulin, total cholesterol, triglyceride, and low-density lipoprotein (P < 0.05) as compared to the controls. Fasting blood glucose and high-density lipoprotein levels were similar between the two groups (P > 0.05). Leptin level in the obese group was also higher (P < 0.05) as compared to that of control group. Results showed that insulin resistance index was positively correlated with body mass index, waist-hip ratio (r = 0.41, P < 0.001; r = 0.245, P < 0.001), and triglyceride level (r = 0.25, P = 0.04). Our study demonstrated that obestatin is negatively correlated with fasting insulin and leptin levels, and positively correlated with insulin resistance in obese children.

Genet. Mol. Res. 2017
DOI: 10.4238/gmr16029210
Research Article

Tetranychus urticae Koch (Acari: Tetranychidae) is considered the main pest of strawberry. Several factors can favor its development, among them the genotype susceptibility and cropping system. The aims of this study were to evaluate the agronomic performance of strawberry cultivars under different managements and to identify strawberry cultivars that meet tolerance to T. urticae and high fruit yield. Thirteen cultivars of strawberry ('Albion', 'Aleluia', 'Aromas', 'Camarosa', 'Camino Real', 'Campinas', 'Diamante', 'Dover', 'Festival', 'Seascape', 'Toyonoka', 'Tudla', and 'Ventana') under three managements (open field, low tunnel, and high tunnel) were evaluated. The T. urticae attack to different cultivars was influenced by managements, being low tunnel the one that provided higher infestations in the most evaluated cultivars. 'Camarosa' was the cultivar with the lower incidence of pest and 'Dover' had the higher infestation. The genotype most suitable for growing under different managements is the 'Festival' genotype, since it meets tolerance to T. urticae, high fruit yield, and phenotypic stability.

Genet. Mol. Res. 2017
DOI: 10.4238/gmr16029599
Research Article

Sexually transmitted infections are an important cause of morbidity among sexually active women worldwide, and have been implicated as cofactors in the pathogenesis of cervical cancer. We investigated the prevalence of human papillomavirus (HPV), Chlamydia trachomatis (CT), and Trichomonas vaginalis (TV), and accessed the diversity of HPV in women with normal and abnormal cytology in Manaus, Brazil. We used polymerase chain reaction and HPV genotyping by direct sequencing. The chi-square test was used to calculate the absolute and relative frequencies of the categorical variables, and Fisher's test was used when P < 0.05. The level of significance was set at 5%. All statistical analyses were performed using R 2.9.0. There were statistically significant differences in age (P = 0.0395), education level (P = 0.0131), sexual partners (P = 0.0211), condom use (P = 0.0039), marital status (P < 0.0001), and pregnancy (P = 0.0003) between the normal and abnormal groups. HPV DNA was found in 36.56 and 93.88% of subjects in the normal and abnormal groups, respectively. A total of 19 genotypes were detected; HPV16 was the most common, followed by HPV58. The percentages of TV and CT DNA were 18.04 and 9.02% in the normal group, respectively. The percentages of HPV/TV and HPV/CT coinfection were 12.5% each in women with normal cytology. These findings improve our understanding of HPV, CT, and TV, and the distribution of HPV types, which may be relevant to vaccination strategies for protecting women from the north of Brazil from cervical cancers and precancerous lesions.

Genet. Mol. Res. 2017
DOI: 10.4238/gmr16029626
Research Article

The use of next-generation sequencing (NGS) technologies provides a great volume of genome sequence data even for non-model species. The development of microsatellite markers using these data is a relatively quick and easy process. Dipteryx alata Vogel (Fabaceae) is an arboreal species from the Cerrado biome and is considered an important plant genetic resource. Here, we report the development of microsatellite markers for D. alata using NGS data. DNA samples from four individuals were sequenced using the Illumina MiSeq platform and high-quality reads were assembled into contigs of the D. alata genome sequence. Microsatellite regions were identified using the IMEX webserver and primer pairs were designed using the Primer3 software. The amplification settings for each locus were optimized. Fluorescent-labeled primers were developed and used to genotype individuals derived from three natural populations of D. alata. Fifty-four microsatellite regions were identified, from which 27 were elected to primer design. Among the amplified loci, 11 were polymorphic, with the number of alleles ranging from 2 to 10. The expected heterozygosity under Hardy-Weinberg Equilibrium (HWE) per locus varied from 0.191 to 0.807. Genotype and allele frequencies for all loci agreed with those expected under HWE and linkage disequilibrium was not significant for all pairs of loci. The probabilities of exclusion of paternity and of combined identity were equal to 0.993 and 5.65 x 10, respectively. The markers developed in this study are useful to several types of population genetic studies with D. alata and, eventually, for closely related species.

Genet. Mol. Res. 2017
DOI: 10.4238/gmr16029639
Research Article

The use of Y chromosome haplotypes, important for the detection of sexual crimes in forensics, has gained prominence with the use of databases that incorporate these genetic profiles in their system. Here, we optimized and validated an amplification protocol for Y chromosome profile retrieval in reference samples using lesser materials than those in commercial kits. FTA cards (Flinders Technology Associates) were used to support the oral cells of male individuals, which were amplified directly using the SwabSolution reagent (Promega). First, we optimized and validated the process to define the volume and cycling conditions. Three reference samples and nineteen 1.2 mm-diameter perforated discs were used per sample. Amplification of one or two discs (samples) with the PowerPlex Y23 kit (Promega) was performed using 25, 26, and 27 thermal cycles. Twenty percent, 32%, and 100% reagent volumes, one disc, and 26 cycles were used for the control per sample. Thereafter, all samples (N = 270) were amplified using 27 cycles, one disc, and 32% reagents (optimized conditions). Data was analyzed using a study of equilibrium values between fluorophore colors. In the samples analyzed with 20% volume, an imbalance was observed in peak heights, both inside and in-between each dye. In samples amplified with 32% reagents, the values obtained for the intra-color and inter-color standard balance calculations for verification of the quality of the analyzed peaks were similar to those of samples amplified with 100% of the recommended volume. The quality of the profiles obtained with 32% reagents was suitable for insertion into databases.

Genet. Mol. Res. 2017
DOI: 10.4238/gmr16029652
Research Article

The aim of the present study is to explore the effect of IL-6 gene polymorphisms on the development of keloid scar (KS) in the Chinese Han population. Genotyping of IL-6 was performed by the polymerase chain reaction (PCR), followed by restriction fragment length polymorphism assays (PCR-RFLP). Serum level of IL-6 was measured using enzyme-linked immunosorbent assay (ELISA). Results indicated that when the IL-6 -572 CC homozygote genotype was used as the reference group, the GG genotype was found to be associated with a significantly increased risk of KS (GG vs CC: OR = 2.097, 95%CI  = 1.100-3.995, P  =  0.025). When the IL-6 -572 C allele was used as the reference group, the G allele was found to be associated with significantly increased risk of KS (G vs C: OR =  1.317, 95%CI  = 1.002-1.730, P = 0.048). Furthermore, we observed a marked increase in serum IL-6 levels in KS patients with GG genotypes when compared to KS patients harboring the CC genotype. In conclusion, our results suggest that IL-6 gene polymorphism was associated with keloid scars in the southeastern Chinese Han population.

Genet. Mol. Res. 2017
DOI: 10.4238/gmr16029110
Research Article

In chronic myeloid leukemia (CML) two main types of messenger RNA (e14a2 and e13a2) can be produced by BCR-ABL1 gene rearrangement. Due to conflicting results, the clinical value of these transcripts remains controversial. The aim of this study was to identify associations of e14a2 and e13a2 transcripts with laboratory variables and also the response to treatment. This study included 203 adult patients with CML treated with Imatinib as first-line drug in a reference hematology center in Northeast Brazil. Clinical and laboratory data were obtained after informed consent. Samples were collected for RNA extraction and analyzed by reverse transcription-polymerase chain reaction (PCR), according to the international protocol BIOMED-1. The LeukemiaNet 2013 criteria were used to establish the molecular response. The frequency distribution of the BCR-ABL1 transcripts was e14a2 (64%), e13a2 (34%), and double positives (2%). The results showed a statistically significant association of the e14a2 transcript type with thrombocytosis (P = 0.0005) and the e13a2 with higher leukocyte count (P = 0.0491). In a subgroup of 44 patients, the molecular response to treatment with Imatinib was assessed by quantitative PCR at 3 months (BCR-ABL1 ≤ 10%), 6 months (BCR-ABL1 ≤ 1%), or 12 months (BCR-ABL1 ≤ 0.1%). Although patients with the transcript e14a2 showed higher frequency of good responses than patients with the transcript e13a2, this difference was not statistically significant. In agreement with published data, our results showed association of the BCR-ABL1 transcript e14a2 with thrombocytosis and the BCR-ABL1 transcript e13a2 with higher leukocytosis in patients with chronic myeloid leukemia.

Genet. Mol. Res. 2017
DOI: 10.4238/gmr16029541

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