Distribution of the Beta-Casein Gene Variants in Three Cattle Breeds Reared in Benin

The beta-casein gene is one of the most functional genetic candidate that affect milk quality and composition traits. Among its variants, the A1/A2 are the most common. Therefore, the aim of this study was to identify the distribution of the Beta-casein gene variants (A1/A2) in three different cattle breeds in order to determine which of the breed produce a better milk for consumers’ health. 152 blood samples which comprises 72 (Muturu), 40 (Azawak) and 40 Girolando were used to carry out this study. Genomic DNA was extracted from the blood samples and each variant was subsequently amplified from the extracted DNA samples using an Allele-Specific PCR technique and then confirmed by running the PCR products on 1% agarose gel. The result showed that there were three genotypes (A1A1, A2A1 and A2A2) in the three breeds. The average percentage genotypic frequencies obtained from this study were 42.76%, 31.58% and 25.66% respectively for A1A1, A1A2 and A2A2 genotypes while the percentage allelic frequencies were 58% and 42% respectively for A1 and A2 allele. The genetic parameters of Azawak breed were higher than that of the other breeds, what implies that there was a higher polymorphism and genetic diversity in the Azawak breed in the beta-casein gene compare to the other breeds. The A2 beta-casein variant in milk has been found to be desirable for milk consumer’s health and nutrition. This study therefore showed that the Azawak breed provides a good potential for increasing this favorable allele through appropriate breeding techniques of cattle.

General Taq PCR Master Mix -- CHEM 384/584 v2

2X PCR Master Mixes are convenient to use as they include all the necessary PCR components except the template and primers. Most PCR Master Mixes also include agarose gel running dyes and a density reagent that allows direct loading of PCR products on an agarose gel for electrophoresis. The master mix format simplifies workflows and sample handling; simply add primers, template, and water and then begin PCR.

Long range PCR-based deep sequencing for haplotype determination in mixed HCMV infections

Background Short read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. Novel third-generation sequencing platforms offer an extended read length and promise to resolve how distant polymorphic sites along individual genomes are linked. In the present study, we established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections. Results Total substitution and indel error rate of mapped reads ranged from 0.17 to 0.43% depending on the stringency of quality trimming. Artificial HCMV DNA mixtures were correctly determined down to 1% abundance of the minor DNA source when the total HCMV DNA input was 4 × 104 copies/ml. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from patient samples. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Alignments of distinct haplotype sequences within patient samples showed uneven distribution of sequence diversity, interspersed by long identical stretches. Moreover, diversity estimation at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed haplotype populations. Conclusions Quantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

Tenebrio molitor: possible source of polystyrene-degrading bacteria

Background The excessive use of polystyrene as a packaging material has resulted in a rise in environmental pollution. Polystyrene waste has continually increased water pollution, soil pollution and the closing of landfill sites since it is durable and resistant to biodegradation. Therefore, the challenge in polystyrene disposal has caused researchers to look for urgent innovative and eco-friendly solutions for plastic degradation. The current study focuses on the isolation and identification of bacteria produced by the larvae of beetle Tenebrio molitor (yellow mealworms), that enable them to survive when fed with polystyrene foam as their sole carbon diet. Materials and methods The biodegradation of polystyrene by Tenebrio molitor was investigated by breeding and rearing the mealworms in the presence and absence of polystyrene. A comparison was made between those fed with a normal diet and those fed on polystyrene. The mealworms which were fed with polystyrene were then dissected and the guts were collected to isolate and identify the bacteria in their guts. The viability and metabolic activity of the isolates were investigated. The polymerase chain reaction (PCR) followed by sequencing was used for molecular identification of the isolates. The PCR products were directly sequenced using Sanger’s method and the phylogenetic tree and molecular evolutionary analyses were constructed using MEGAX software with the Neighbour Joining algorithm. The evolutionary distances were computed using the Maximum Composite Likelihood method. Results The decrease in mass of the polystyrene as feedstock confirmed that the mealworms were depending on polystyrene as their sole carbon diet. The frass egested by mealworms also confirmed the biodegradation of polystyrene as it contained very tiny residues of polystyrene. Three isolates were obtained from the mealworms guts, and all were found to be gram-negative. The sequencing results showed that the isolates were Klebsiella oxytoca ATCC 13182, Klebsiella oxytoca NBRC 102593 and Klebsiella oxytoca JCM 1665. Conclusion Klebsiella oxytoca ATCC 13182, Klebsiella oxytoca NBRC 102593 and Klebsiella oxytoca JCM 1665 maybe some of the bacteria responsible for polystyrene biodegradation.

General Taq PCR Master Mix -- CHEM 384/584 v2

SapphireAmp Fast PCR Master Mix contains a hot start PCR enzyme, optimized buffer, dNTP mixture, gel loading dye (blue), and a density reagent as a 2X premix. SapphireAmp Fast PCR Master Mix is optimized for fast PCR and offers a rapid extension rate (10 sec. per kb). The inclusion of blue dye and a density reagent allows direct loading of PCR products on an agarose gel for electrophoresis. The master mix format simplifies workflows and sample handling; simply add primers, template, and water and then begin PCR. SapphireAmp Fast PCR Master Mix is ideal for fast colony PCR screening. Fast colony PCR amplification of a 5 kb insert can be completed in approximately 1 hr 15 min. Furthermore, it is possible to amplify fragments up to 6 kb from genomic DNA templates.

Diagnosis and detection of VicK gene in Streptococcus mutans isolated from the saliva of patients with diabetic type 2 with tooth decay in the Iraqi population

Type 2 diabetes mellitus (T2DM) is gradually becoming more common in Iraq. Salivary changes and proliferation of specific bacterial communities cause oral disease that can adversely affect systemic conditions such as diabetes. Fifty saliva samples were collected from people with T2DM suffering from tooth decay and twenty-five people without T2DM suffering from tooth decay. The periodontal status, the extent of the root surface, and coronal caries were evaluated. Saliva was cultured for investigating Streptococcus mutans. The results showed that patients with type 2 diabetes had significantly more severe Periodontitis and a higher prevalence and magnitude of bacterial caries. Diabetic subjects had higher levels of Hemoglobin A1c (HbA1c) and Random Blood Sugar (R.B.S.). The S. mutans diagnosis by PCR for Sanger Sequencing technique by using VicK gene sequences (1300bp). The PCR products of the isolate were submitted to Macrogen Company for sequencing. Selected seven isolates as new isolates registered in global gene bank as locally S. Mutans isolates in Bagdad city/Iraq and their accepted accession numbers include LOCUS MT603520, MT603521, MT603522, MT603523, MT603524, MT603525,and MT603526 of nucleotide sequence. The VicK genes isolates' phylogenetic trees revealed a genotype that was closely connected to other isolates in GenBank. Furthermore, gene sequencing demonstrated a success rate of 99 percent. resemblance to other isolates in the GenBank database The likelihood of a link between S. Mutans and dental carries was determined by these findings.

Yersinia massiliensis (Enterobacteriales: Enterobacteriaceae) in the host Anaphes nitens (Hymenoptera: Mymaridae): first report of association with insects

Endosymbiont bacteria can affect biological parameters and reduce the effectiveness of natural enemies in controlling the target insect. The objective of this work was to identify endosymbiont bacteria in Anaphes nitens (Girault, 1928) (Hymenoptera: Mymaridae), the main natural enemy used to manage Gonipterus platensis (Marelli, 1926) (Coleoptera: Curculionidae). Genomic DNA from six A. nitens populations was extracted and polymerase chain reactions (PCR) were performed with the primers to detect endosymbiont bacteria in this insect. The PCR products were amplified, sequenced, and compared with sequences deposited in the GenBank for the bacteria identification. All A. nitens populations had the bacterium Yersinia massiliensis (Enterobacteriales: Enterobacteriaceae). This bacterium was originally described as free-living, and it is associated with and composes part of the A. nitens microbiota. This is the first report of Y. massiliensis in an insect host.

Identification of 19-bp indel of the Pleomorphic Adenoma Gene 1 in Bali cattle population

Pleomorphic adenoma gene 1 (PLAG1) is a zinc finger transcription factor gene located on bovine chromosome 14 (BTA14) affecting body size and reproduction traits in cattle. The objective of this study was to identify 19-bp indel of the PLAG1 gene in Bali cattle population. A total of 96 blood samples of Bali Cattle were collected from Balai Pembibitan Ternak Unggul dan Hijauan Pakan Ternak (BPTU-HPT) Denpasar. Genomic DNA was extracted from blood samples and used to detect 19-bp indel of the PLAG1 gene using following primer pair 5’-TCCGAACAACAGGTGAGGGAGAAAT-3’ and 5’-CCACTTCAGG-GGTGCTCTAGGTTTG-3’. The polymerase chain reaction (PCR) products using DNA pool samples were sequenced to validate the PCR product and to find out novel polymorphism in Bali cattle population. The result showed that there was no variation found in Bali cattle population based on 19-bp indel of the PLAG1 gene, which is indicated by 123 bp DNA band. However, sequence analysis of the PLAG1 gene resulted in a novel single nucleotide polymorphism (SNP) at nucleotide number 32235 of the PLAG1 gene that changed guanine (G) to adenine (A). This novel SNP could be furthermore genotyped and it might be a potential candidate marker for body size and reproduction traits in Bali cattle.

First Detection of Lake Sinai Virus in the Czech Republic: Possibility of Novel LSV Species

Lake Sinai virus (LSV) is one of more than twenty honeybee viruses. There are officially two species: LSV 1 and LSV 2. However, there is currently a limited number of whole-genome sequences and the genetic variability of the virus indicates more than two species exist. Extracted nucleic acid of honeybee samples were screened by PCR for presence of the selected honeybee viruses. LSV was the third most abundant virus (36.9% of positive samples) after Apis mellifera filamentous virus (72.2%) and Deformed wing virus (52.5%). LSV-positive samples underwent additional PCR reaction with primers targeting region coding RNA-dependent RNA polymerase of the virus. The PCR products were sequenced and the acquired sequences used for first phylogenetic analysis. Based on the results, several of the isolates were selected to undergo whole-genome sequencing and the sequences obtained were used for additional phylogenetic analyses and construction of dendrograms. The results indicate presence of at least three genetically distinct groups of LSV in the Czech Republic, the major one being related to LSV 2, but too distinct to be considered LSV 2 species. Two sequences of major Czech LSV cluster of strains were successfully acquired, thus being the first Czech LSV strains published to date.

POLYMORPHISM OF TAGLGAP MICROSATELITE LOCUS AND ITS CONNECTION WITH ALLELELIC VARIETIES OF GLIADINS OF BREAD WHEAT

Introduction. Gliadins are monomeric and highly polymorphic storage proteins of wheat endosperm, which together with glutenins form a gluten complex that determines the breadmaking properties of wheat. Allelic variants of gliadins are an important feature in the selection of material for breeding, but their determination by electrophoresis in acid PAGE is quite difficult. Aim. The aim of this study was to investigate the polymorphism of the Taglgap microsatellite locus and to analyze its correspondence to the polymorphism of allelic variants of gliadins that have been revealed by acid PAGE electrophoresis. Methods. 140 cultivars and lines of bread wheat of Ukrainian and foreign selection were analyzed. Electrophoresis of storage proteins was performed in an acid PAGE according to the method of F. O. Poperellia (1989), allelic variants were designated according to the international nomenclature (Metakovsky et al., 2018). DNA was isolated by CTAB method and PCR was performed with primers to the Taglgap microsatellite (Devos et al., 1995). PCR products were fractionated in 7% PAGE and stained with silver staining method. Nucleotide sequences were searched by BLAST and aligned by MAFT methods. The main results. 19 allelic variants of gliadins and 11 alleles of the Taglgap locus were identified. In the collection of Ukrainian varieties there were Gli-B1b, Gli-B1c, Gli-B1d, Gli-B1e, Gli-B1f, Gli-B1g, Gli-B1h, Gli-B1l and Gli-B1o allelic variants and alleles of Taglgap 216 bp, 237 bp, 246 bp, 248 bp, 252 bp, 267 bp, 270 bp and null. In the foreign collection of varieties − Gli-B1a, Gli-B1b, Gli-B1c, Gli-B1d, Gli-B1e, Gli-B1f, Gli-B1g, Gli-B1h, Gli-B1i, Gli-B1j, Gli-B1k, Gli -B1l, Gli-B1m, Gli-B1n, Gli-B1o, Gli-B1p, Gli-B1q, Gli-B1r, Gli-B1s and 213 bp, 216 bp, 237 bp, 246 bp, 248 bp, 250 bp, 252 bp, 270 bp, 285 bp and null. Nucleotide sequence analysis in the NCBI database showed the presence of a number of other alleles of the Taglgap microsatellite not only in bread wheat but also in some species of the Triticum L. and Aegilops L. genus. Conclusions. The detected polymorphism correlates with the polymorphism of allelic variants of gliadins of Gli-B1 locus and makes it possible to identify Gli-B1a, Gli-B1d, Gli-B1h and Gli-B1l allelic variants, and for Ukrainian varieties with high probability also Gli-B1b allelic variant. However, this marker does not allow identifying Gli-B1c, which is important for selection.