Table of Contents | Genet. Mol. Res. 2021 (4)
Seed aging is a complex process that includes degradation of macromolecules, such as DNA. Evaluation of DNA deterioration in asymptomatic stages of seed aging may help the development of improved methods for monitoring long-term conserved seeds. We examined DNA integrity in artificially aged soybean (Glycine max) seed lots using cytogenetics, quantitative polymerase chain reaction (qPCR), and random amplified polymorphic DNA (RAPD). Freshly harvested soybean cultivar BRS 7980 seeds were dried and kept at 10oC in a dry room until the analyses. Six lots of 450 seeds were artificially aged (42°C, 100% relative humidity) for 6, 12, 24, 48, 72 or 96 h to obtain lots with different germination capacities. One unaged seed lot (0 h) was used as a control. Germination of aged lots varied from 12 to 86% at 96 and 6 h of aging, respectively. Cytogenetic data did not characterized aged seed. DNA integrity among seed lots was also evaluated based on the comparison of frequencies of three fragments amplified using an anchored variable-length primer set. Fragments of 86 and 193 bp had similar frequencies, while the largest one (491 bp) showed greater variation among samples. Data suggested different fragment frequencies were due to variation in the number of intact templates among lots, which might have been caused due to the fact that shorter DNA sequences have a smaller probability of developing random breaks than larger ones. RAPDs confirmed the differences in template amount detected using qPCR and variation in DNA repair capacity among aged lots. Though some seed lots were characterized using physiological parameters, most of them could not be characterized based on cytogenetic and molecular data, suggesting that these tests might not be ideal to detect aging in soybean seeds.
In silico microsatellite transferability from Psidium guajava to Eucalyptus globulus validated by PCR
BLAST is a genomic local alignment search tool used to identify homology between genotypes and possible orthologous genes. In vitro microsatellite transferability is a strategy to enable or increase species molecular fingerprinting, but it is dependent on PCR technique. An initial in silico step using BLAST for transferability can be helpful to save resources in pre-selecting markers more likely to amplify. We aligned and transfered SSR sequences from Psidium guajava to Eucalyptus globulus using BLAST. Twenty-three SSR clone sequences from P. guajava (query) were retrieved from the NCBI website and aligned against the whole genome of E. globulus (subject) using a cut-off e-value<1.00e−20. Another 140 loci retrieved from the GuavaMap project were analyzed using as parameters e-values<1.7 and a maximum distance of 300 nucleotides between forward and reverse sequences. All loci were analyzed using BLASTN with MEGABLAST optimization. DNA extraction of four eucalypt trees was performed with the 2x CTAB protocol containing a sorbitol initial step. Validation of the SSR selected via BLASTN was performed by PCR reactions with 12 loci (seven selected on Blast hits and five without hits) and posterior visualization on polyacrylamide gel. Nine out of 23 microsatellite loci were transferable in the in silico , with a mean identity of 87%. With regard to the GuavaMap microsatellite loci, only three showed significant alignments, among the 140 tested, with the forward and reverse mean identity of 100% and 95%, respectively. All seven SSR with e-values<1.00e−20 (mPgCIR001, mPgCIR005, mPgCIR007, mPgCIR009, mPgCIR018, mPgCIR020, and mPgCIR026) showed easy-to-score amplicons on the polyacrylamide gel when using the in silico transferability strategy. However, the other five, without significant e-values or hits, showed no amplification. These results highlight the effectiveness of in silico transferability for full-length SSR loci, constituting a valid alternative to save time and costs in transferability studies between species.
Periclinal chimera are made constituted of two genotypes growing side by side. One of these genotypes makes up the epidermis, the second forms the internal tissues. As a nonconventional method to improve cassava, it brought its productivity to an extraordinary level that has never been reported before. In previous experiments the chimera were synthesized by hormone treatment, which was applied to the surface of the grafts to promote callus formation. We propose here a simple method that significantly increases the induction of periclinal chimera. It is principally to make grafts in which scions are cut in a slanted position close to a bud and the rootstock cut in the opposite direction. The scion and the rootstock are placed in close contact, having the juxtaposition of the scion and the rootstock so that the buds can make contact with each other. A cello tape is used to fasten and hold them together. We also interpret what has been noted of exceptional chimera productivity based on combining ability between genotypes of multiple ploidy levels and the movement of DNA from one periclinal chimera layer to another. Manihot fortalizensis showed the highest combining ability and the highest compatibility with all cassava cultivars tested. Before synthesizing periclinal chimera it is recommended to examine combining ability between candidate species and varieties.
Non-synonymous de novo gene mutations in Wilms’ Tumor: Identification and characterization of new variants of WT1 and WT2 loci in Indian Population
Wilms tumor (WT) is a complex pediatric disease involving both genetic and epigenetic factors. There is strong evidence that mutations at WT1 and WT2 loci are associated with the etiopathology of WT cases. In humans, large numbers of predisposition genes are distributed throughout the genome, and their functional aptitude has not been defined clearly in tumor biology. We examined new variants of WT1 and WT2 in WT cases in Eastern India by performing gene analysis using Sanger sequencing. The study was further extended to translate nucleotide variants into the corresponding amino acids to explore the mechanisms of tumorigenesis. Using bioinformatics tools, the WT1 locus showed single nucleotide substitutions in the sequence, i.e. TGT → CGT or ACC→GCC, resulting in changes in amino acids; i.e. arginine is replaced by cysteine or alanine by threonine, suggesting that these changes might either alter zinc finger DNA binding domains or be involved in the synthesis of altered proteins during tumor cell differentiation. Similarly, the WT2 locus showed non-synonymous de-novo gene mutations, CAC→TAA, TAT→TAA, TGA→ GGA, and these new variations correspond to either histidine or tyrosine or glycine, respectively, resulting in failure to regulate transcription, suggesting that these nucleotide sequences can be considered as “stop codons” or pre termination codons. In conclusion, we identified new variants of WT1 and WT2 loci, leading to truncated proteins that may play a role in tumorigenesis in WT cases.
Karyotypic characterization of Symphurus tessellatus and Symphurus plagusia (Pleuronectiformes, Cynoglossidae) from Brazilian coastal waters
The family Cynoglossidae (tonguefishes) is a speciose group of Pleuronectiformes, encompassing about 20% of described species in this order, distributed among three genera (Cynoglossus, Paraplagusia and Symphurus). Symphurus is the only genus of tonguefish in the Western Atlantic, being characterized by species complexes and cryptic forms; consequently the actual species richness of this genus is likely to be underestimated. Comparative cytogenetic studies have proved to be useful to resolve taxonomic uncertainties in ichthyofauna. Therefore, we carried out the karyotypic characterization of Symphurus tessellatus and Symphurus plagusia from the Brazilian coast based on conventional analysis, C-banding, silver nitrate (Ag-NORs) and base-specific fluorochrome staining. The specimens of S. tessellatus presented 2n = 46, 20 m/sm+26st/a, differing from that previously reported (2n = 46, 22m/sm+24 st/a), with heterochromatic blocks in the centromeric region of most chromosomes, as usually described in this group. We also found interstitial segments on the long arms of the third chromosome; these were coincident to Ag-NORs and GC-rich sites. In S. plagusia, the karyotype is composed of 32m/sm+14st/a (2n=46), with a single nucleolar organizer regions (NORs) system, which is the first cytogenetic data for this species. The unique karyotype formulae of these two species suggest that pericentric inversions played a major role in the chromosomal differentiation of tonguefishes, being useful for cytotaxonomy and diagnosis of evolutionary units in Symphurus.
Considering the great economic and social importance of sweet potato and the few transcriptome studies carried out on this species so far, which can potentially lead to significant improvements in its production system at both productivity and quality levels, this study aimed to determine the most suitable methodology for extracting high-quality RNA from sweet potato’s tuberous roots, branches, and leaves. The experiment was composed of three biological replicates, each one comprising three plants. 100 mg of ground tissue was used for isolating RNA through the CTAB and TRIzol methods, while 160 mg was used for the Hot Phenol Acid method. From the three tested protocols, all of them enabled the isolation of RNA at quantities above 250 ng/μL for the three different tissues, which is the minimal quantity required for conducting molecular assays such as RT-qPCR. However, in terms of RNA quality, evaluated through the A260/A280 and A260/A230 ratios, only the Hot Phenol Acid and CTAB methods generated satisfactory results, displaying values from 1.8 to 2.2. To conclude, the hot acid phenol and CTAB methods, not commonly used for transcriptional studies in sweet potato, are excellent choices for the RNA extraction from different sweet potato tissues.
Temporal data series and logistic models reveal the dynamics of SARS-CoV-2 spike protein D614G variant in the COVID-19 pandemic
The COVID-19 pandemic is caused by the worldwide spread of the RNA virus SARS-CoV-2. Because of its mutational rate, wide geographical distribution, and host response variance this coronavirus is currently evolving into an array of strains with increasing genetic diversity. Most variants apparently have neutral effects for disease spread and symptom severity. However, in the viral spike protein, which is responsible for host cell attachment and invasion, the D614G variant, containing the amino acid substitution D to G in position 614, was suggested to increase viral infection capability. Here we propose a novel method to test the epidemiological impact of emergence of a new variant, by a combination of epidemiological curves (for new cases) and the temporal variation of relative frequencies of the variants through a logistic regression model. We applied our method to temporal distributions of SARS-CoV-2 D614 or G614, in two geographic areas: USA (East Coast versus West Coast) and Europe-Asia (East Countries versus West Countries). Our analysis shows that the D614G prevalence and the growth rates of COVID-19 epidemic data curves are correlated at the early stages and not correlated at the late stages, in both the USA and Europe-Asia scenarios. These results show that logistic models can reveal the potential selective advantage of D614G, which can explain, at least in part, the impact of this variant on COVID-19 epidemiology.
Estimate of genetic diversity in germplasm of elephant grass accessions in Brazil using the Gower algorithm
Management of variability in germplasm banks is essential for genetic improvement, so that the breeder can estimate the genetic similarity between cultivars, as well as maintain genetic diversity in breeding programs. Elephant grass is a forage crop plant native to Africa of great socio-economic and environmental importance; it can be used for animal feed and for bioenergy production. Understanding the genetic variability of elephant grass is essential for breeding programs. In this context, we examined the genetic divergence of elephant grass accessions using the Gower algorithm. The experiment was conducted under field conditions in the municipality of Campos dos Goytacazes-RJ, Brazil. All 85 elephant grass accessions belonging to the Active Elephant Grass Germplasm Bank of Embrapa Gado de Leite were included. These genotypes are commercial varieties from various countries. They were evaluated for morphoagronomic, morphological and phenological characteristics. The experimental design was randomized blocks with two repetitions. The plots were composed of 5.5-meter rows, with 2.0-meter spacing between the planting rows, totaling 11.0 m2. The useful area was a sample in the center of the plot. We generated an illustrative dendrogram, obtained by the UPGMA method and the Tocher clustering, based on the Gower algorithm. Data were examined by means of the GENES statistical programs and the R program. According to the dissimilarity matrices based on the Gower algorithm, the genetic distances varied between 0.08 and 0.56, and the mean distance of the 85 evaluated accessions was 0.25, suggesting, consequently, that there is wide genetic variability between the accessions. Of the 85 genotypes, seven presented genetic distances smaller than 0.1, being indicative of duplicates in the germplasm bank, which could be eliminated without risk of loss of genetic variability.
The application in a single or split dose of nitrogen in wheat affects the expression of ear components. Models that simulate these effects can help predict yield. Our objective was to identify wheat ear components that are responsive to the forms of nitrogen supply in a single versus split dose d. This was achieved by testing variables in the construction of a multiple linear regression model for grain yield simulation in harvest simulation analysis. The study was carried out in 2018 and 2019, in a randomized block design with four replications in a 3 x 3 factorial scheme, for three doses of nitrogen (30, 60, 120 kg ha-1) and three forms of nutrient supply [ single dose (100%) at phenological stage V3 (third expanded leaf); split dose (70% and 30%) at phenological stage V3/V6 (third and sixth expanded leaf) and split dose (70% and 30%) at phenological stage V3/R1 (third expanded leaf and beginning of grain filling)] , respectively, in the soybean/wheat and corn/wheat succession systems. This form of fractionation of 70% and 30% is the form commonly used in commercial wheat crops. The wheat cultivar used was BRS Guamirim. Twenty ears of wheat per experimental unit were randomly collected, which were sent to the laboratory. Ear mass (EM, g), ear grain mass (EGM, g), ear grain number (EGN, n), ear length (EL, cm) and ear harvest index (EHI), given by the ratio of ear grain mass to ear mass, dry weight were measured. Nitrogen supplied in single and split doses modifies with greater intensity the wheat ear components ear mass and ear grain mass. The nitrogen in a single dose supplied at stage V3 provides significant superior grain yield compared to split doses, regardless of nutrient dose, crop season and succession system.
Morphological and anatomical characterization of peduncle, flower and fruit related to easy fruit abscission of Capsicum chinense (Solanaceae) genotypes
The sweet and chili pepper species of the genus Capsicum are originally from America; however, currently they are grown worldwide. Although widely appreciated, especially in the Amazon region, there have been few studies about C. chinense. Characterization and classification studies can aid in pepper crop genetic breeding and development of new cultivars with characteristics of agronomic interest. Resistance to fruit abscission makes harvesting difficult, which today is essentially manual, so that development of cultivars with easy fruit abscission, allowing semi-mechanized and mechanized harvest is an important goal. Descriptions of genetic differences based on anatomical characters are rare in this genus. Along this line, we examined morphological characters and anatomical structures directly related to easy fruit abscission in C. chinense, through the evaluation of two genotypes contrasting for this character, maintained by the Embrapa Hortaliças Genetic Breeding Program, as well as an F1 hybrid. The morphological characterization of flowers and fruits was performed using 13 descriptors, eight defined by the International Plant Genetic Resources Institute and five adapted for this study. The anatomical characterization of the peduncle and flowers was carried out with the elaboration of plant slides to examine structures possibly linked to easy abscission of the fruit. Contrasting morphological polymorphisms were observed in immature fruit color, fruit shape, fruit position on the plant, abscission and fruit firmness, as well as fruit diameter, number of locules and seeds per loculus. Small differences were observed in the anatomical structures of peduncle and flower, such as the shape of the vascular system and lignification of the pericycle, but none were directly related to fruit abscission. Although there was no significant anatomical relationship with fruit abscission, the morphological characters evaluated in this study revealed great genetic variability for these genotypes, demonstrating potential of these genotypes in a breeding program for developing varieties with easy fruit abscission.
The species Nicotiana tabacum, known as tobacco, is one of the crops with the highest economic value in the world among non-food species. Since 2000, Brazil has become the world’s second largest tobacco producer. Brazilian production is mainly concentrated in the South region. The main tobacco types are produced in Brazil, such as Dark, which, despite representing a small percentage of the total production in Brazil, is economically important because of a high added value. Until now the only available cultivar is a line that despite having a desirable alkaloid content, has low productivity. In an effort to help improve production, we estimated the heterosis of diallel crosses involving tobacco lines of the Dark tobacco type. A complete diallel cross was made involving 10 lines from the British American Tobacco company breeding program. The 45 hybrid combinations, together with the 10 parental lines and nine other commercial controls, were evaluated at four sites in Southern Brazil, using a triple lattice experimental design (8 x 8). The characteristics evaluated included: green leaf mass (Productivity - YLD), and total alkaloid content (ALK), and the selection index was obtained by the sum of standardized variables (SSV), considering a weight of 70% for YLD and 30% for ALK. Estimated correlation between YLD and the ALK was -0.66. The use of the selection index allowed simultaneous gains in both traits, although they were of less magnitude than selection for each characteristic individually. The average heterosis was 8.6% for productivity, -1.4% for the total alkaloid content, and 5.3% for the selection index. However, considering the selection index, hybrids were obtained with an average heterosis greater than 10%. Under these conditions, the use of hybrids should be encouraged not only to combine favorable phenotypes, but also to explore the beneficial effects of heterosis.
Carrot is a vegetable of worldwide importance due to its high nutritional quality and wide application in food. The adjustment of plant arrangement in carrot cultivars is decisive for the productivity and quality of roots; in a conventional system, a spacing between rows of 20 cm is indicated. However, few studies address the interaction cultivar versus row spacing in an agro-ecological cropping system, suitable for production without pesticides and fertilizers. We examined the performance of carrot cultivars at different spacing between rows on the yield and quality of roots in an agro-ecological system, with the objective of greater sustainability of food security cultivation. This experiment was carried out in Augusto Pestana, RS, Brazil, from February to June, in 2019 and 2020. The design was a randomized block with three replications in a 5x3 factorial scheme, for five carrot cultivars (Danvers, Brasília Calibrada Media, Nantes, Esplanada and Brasília Nina) and three spacings between lines (10, 15 and 20 cm), respectively. The cultivars used are the most used accepted genotypes for cultivation in Brazil. In the study, the indicators of productivity and quality of roots were evaluated. The Brasília Nina carrot cultivar showed high performance in root yield and quality, mainly in the 15 cm spacing between rows in an agro-ecological system. The production of carrot roots with quality and free from toxic contaminants, contributes to the valorization of the product for commercialization and the production of food with greater sustainability and food safety.