Single Nucleotide Polymorphism Patterns Research Articles

Single Nucleotide Polymorphism-based Identification of Bacterial Artificial Chromosome-mediated Homologous Recombination.

Bacterial Artificial chromosome (BAC) recombineering is a powerful genetic manipulation tool for the efficient development of recombinant genetic resources. Long homology arms of more than 150 kb composed of BAC constructs not only substantially enhance genetic recombination events, but also provide a variety of single nucleotide polymorphisms (SNPs) that are useful markers for accurately docking BAC constructs at target sites. Even if the BAC construct is homologous to the sequences of the target region, different variations may be distributed between various SNPs within the region and those within the BAC construct. Once the BAC construct carrying these variations was precisely replaced in the target region, the SNP profiles within the target genomic locus were directly replaced with those in the BAC. This alteration in SNP profiles ensured that the BAC construct accurately targeted the designated site. In this study, we introduced restriction fragment length polymorphism or single-strand conformation polymorphism analyses to validate and evaluate BAC recombination based on changes in SNP patterns. These methods provide a simple and economical solution to validation steps that can be cumbersome with large homologous sequences, facilitating access to the production of therapeutic resources or disease models based on BAC-mediated homologous recombination.

TNFα rs1800629 Polymorphism and Response to Anti-TNFα Treatment in Behçet Syndrome: Data from an Italian Cohort Study.

Tumor Necrosis Factor-alpha (TNFα) rs1800629 (-308G>A) is a single nucleotide polymorphism (SNP) related to variable responses to anti-TNFα therapy. This therapy is efficient in severe and refractory manifestation of Behçet syndrome (BS), an auto-inflammatory systemic vasculitis. We investigated (1) the association between rs1800629 genotypes and responses to therapy and (2) the correlation between SNP and clinical patterns in a cohort of 74 BS Italian patients receiving anti-TNFα therapy with a follow-up of at least 12 months. The rs1800629 was genotyped through amplification, direct sequencing and bioinformatics analyses. The rs1800629 GG and GA genotypes were assessed as predictors of outcomes dividing the patients between therapy responders and non-responders. The rs1800629 GG and GA genotypes were found, respectively, in 59/74 (79.7%) and 15/74 BS patients (21.3%) (p < 0.05). We identified 16/74 (21.9%) non-responder patients, of which 9/16 (56.3%) showed the GG genotype and 7/16 (43.7%) the GA genotype. A total of 50/58 (86.2%) responder patients showed the GG genotype, and 8/58 (13.8%) the GA genotype (p < 0.05). The percentage of non-responder females (68.8%) was significantly higher than non-responder males (31.2%) (p < 0.05). No correlation between SNP and clinical patterns was observed. To successfully include rs1800629 as a predictive biomarker of TNFα inhibitor response, genome-wide association studies in larger, well-characterised cohorts are required.

Going beyond consensus genome sequences: An innovative SNP-based methodology reconstructs different Ugandan cassava brown streak virus haplotypes at a nationwide scale inRwanda.

Cassava Brown Streak Disease (CBSD), which is caused by cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), represents one of the most devastating threats to cassava production in Africa, including in Rwanda where a dramatic epidemic in 2014 dropped cassava yield from 3.3 million to 900,000 tonnes (1). Studying viral genetic diversity at the genome level is essential in disease management, as it can provide valuable information on the origin and dynamics of epidemic events. To fill the current lack of genome-based diversity studies of UCBSV, we performed a nationwide survey of cassava ipomovirus genomic sequences in Rwanda by high-throughput sequencing (HTS) of pools of plants sampled from 130 cassava fields in thirteen cassava-producing districts, spanning seven agro-ecological zones with contrasting climatic conditions and different cassava cultivars. HTS allowed the assembly of a nearly complete consensus genome of UCBSV in twelve districts. The phylogenetic analysis revealed high homology between UCBSV genome sequences, with a maximum of 0.8 per cent divergence between genomes at the nucleotide level. An in-depth investigation based on Single Nucleotide Polymorphisms (SNPs) was conducted to explore the genome diversity beyond the consensus sequences. First, to ensure the validity of the result, a panel of SNPs was confirmed by independent reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. Furthermore, the combination of fixation index (FST) calculation and Principal Component Analysis (PCA) based on SNP patterns identified three different UCBSV haplotypes geographically clustered. The haplotype 2 (H2) was restricted to the central regions, where the NAROCAS 1 cultivar is predominantly farmed. RT-PCR and Sanger sequencing of individual NAROCAS1 plants confirmed their association with H2. Haplotype 1 was widely spread, with a 100 per cent occurrence in the Eastern region, while Haplotype 3 was only found in the Western region. These haplotypes' associations with specific cultivars or regions would need further confirmation. Our results prove that a much more complex picture of genetic diversity can be deciphered beyond the consensus sequences, with practical implications on virus epidemiology, evolution, and disease management. Our methodology proposes a high-resolution analysis of genome diversity beyond the consensus between and within samples. It can be used at various scales, from individual plants to pooled samples of virus-infected plants. Our findings also showed how subtle genetic differences could be informative on the potential impact of agricultural practices, as the presence and frequency of a virus haplotype could be correlated with the dissemination and adoption of improved cultivars.

Evaluation of SNP-Based Markers Utilization for Resistance to Fall Armyworm Spodoptera frugiperda on Eight Corn Varieties

Abstract The aim of the study was to compare responses of different corn varieties to the fall armyworm Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae) attack and to map patterns of single nucleotide polymorphism of the RGA genes. The study consisted of two activities, i.e., the field experiment to observe corn damage intensity and the laboratory observation to study the SNP pattern. Field experiment was conducted to compare eight corn varieties set in randomized block design with 3 replications. Damage intensity of the corn due to S. frugiperda attack was observed on the field and the corn samples were analyzed in the laboratory to examine the RGA (resistance gen analogue) which contained single nucleotide polymorphism (SNP). The results from the field and the laboratory analysis showed that the response of Bisi 321 variety to S. frugiperda attack was better than the other tested varieties. Based on the electrophoresis of the PCR results with the SNP2_MNBS_Alt primer, samples of corn varieties coded as V1 (Pertiwi 5), V3 (P-36), V6 (NK Super), V7 (Exotic), and V8 (Local) produced amplicons with the size of ±150 bp. The samples that did not have amplification were V2 (NK7328), V4 (Bisi 321), and V5 (Bisi 18). There is a possibility that the amplification results may indicate some degrees of tolerance clusters of corn varieties to S. frugiperda attack. Further validation is required to apply the SNP-based markers of corn to develop tolerant or resistant varieties. Keywords: Spodoptera frugiperda, RGA, SNP, Corn

Performance of the tetra-primer PCR technique compared to PCR-RFLP in the search for rs12979860 (C/T) and rs8099917 (T/G) single nucleotide polymorphisms (SNPs) in the IFNL4 gene

Single nucleotide polymorphisms (SNPs, rs12979860 e rs8099917) in the Interferon Lambda 4 gene (IFNL4, formerly IFNL3 and/or IL28B) has been associated with failure in the innate immune response, sustained virological response in hepatitis C, and HTLV-1-associated myelopathy (HAM) development. To search for these polymorphisms several methodologies can be employed, such as sequencing, real-time or quantitative polymerase chain reaction (qPCR), restriction fragment length polymorphism analysis in PCR products (PCR-RFLP), and tetra-primer PCR. The present study compared the performance of the tetra-primer PCR in relation to the PCR-RFLP, both optimized in the Research HTLV Laboratory of the Center of Immunology of Instituto Adolfo Lutz in São Paulo. One hundred DNA samples obtained from patients of STD/Aids Reference Centre in São Paulo, previously analyzed for IL28B SNPs by PCR-RFLP were selected for analysis, after confirming that they represent all IL28B SNPs patterns described in the literature. The results obtained showed concordance between the PCR-RFLP and the tetra-primer PCR SNPs results, and because of the low cost, easy to perform, and minor employment of biological specimen and reagents, the tetra- primer PCR is of choice to be used in routine.

Performance of the tetra-primer PCR technique compared to PCR-RFLP in the search for rs12979860 (C/T) and rs8099917 (T/G) single nucleotide polymorphisms (SNPs) in the IFNL4 gene

Single nucleotide polymorphisms (SNPs, rs12979860 e rs8099917) in the Interferon Lambda 4 gene (IFNL4, formerly IFNL3 and/or IL28B) has been associated with failure in the innate immune response, sustained virological response in hepatitis C, and HTLV-1-associated myelopathy (HAM) development. To search for these polymorphisms several methodologies can be employed, such as sequencing, real-time or quantitative polymerase chain reaction (qPCR), restriction fragment length polymorphism analysis in PCR products (PCR-RFLP), and tetra-primer PCR. The present study compared the performance of the tetra-primer PCR in relation to the PCR-RFLP, both optimized in the Research HTLV Laboratory of the Center of Immunology of Instituto Adolfo Lutz in São Paulo. One hundred DNA samples obtained from patients of STD/Aids Reference Centre in São Paulo, previously analyzed for IL28B SNPs by PCR-RFLP were selected for analysis, after confirming that they represent all IL28B SNPs patterns described in the literature. The results obtained showed concordance between the PCR-RFLP and the tetra-primer PCR SNPs results, and because of the low cost, easy to perform, and minor employment of biological specimen and reagents, the tetra- primer PCR is of choice to be used in routine.

Improving efficiency of fitting Cox proportional hazards models for time-to-event outcomes in genome-wide association studies (GWAS).

Technologies identifying single nucleotide polymorphisms (SNPs) in DNA sequencing yield an avalanche of data requiring analysis and interpretation. Standard methods may require many weeks of processing time. The use of statistical methods requiring data sorting, matrix inversions of a high-dimension and replication in subsets of the data on multiple outcomes exacerbate these times.A method which reduces the computational time in problems with time-to-event outcomes and hundreds of thousands/millions of SNPs using Cox-Snell residuals after fitting the Cox proportional hazards model (PH) to a fixed set of concomitant variables is proposed. This yields coefficients for SNP effect from a Cox-Snell adjusted Poisson model and shows a high concordance to the adjusted PH model.The method is illustrated with a sample of 10000 SNPs from a genome-wide association study in a diabetic population. The gain in processing efficiency using the proposed method based on Poisson modelling can be as high as 62%. This could result in saving of over three weeks processing time if 5 million SNPs require analysis. The method involves only a single predictor variable (SNP), offering a simpler, computationally more stable approach to examining and identifying SNP patterns associated with the outcome(s) allowing for a faster development of genetic signatures. Use of deviance residuals from the PH model to screen SNPs demonstrates a large discordance rate at a 0.2% threshold of concordance. This rate is 15 times larger than that based on the Cox-Snell residuals from the Cox-Snell adjusted Poisson model. The method is simple to implement as the procedures are available in most statistical packges. The approach involves obtaining Cox-Snell residuals from a PH model, to a binary time-to-event outcome, for factors which need to be common when assessing each SNP. Each SNP is then fitted as a predictor to the outcome of interest using a Poisson model with the Cox-Snell as the exposure variable.

Allelic Variants of HLA-C Upstream Region, PSORS1C3, MICA, TNFA and Genes Involved in Epidermal Homeostasis and Barrier Function Influence the Clinical Response to Anti-IL-12/IL-23 Treatment of Patients with Psoriasis.

Several biologic therapies have been developed to treat moderate-to-severe psoriasis, with patients exhibiting different clinical benefits, possibly due to the heterogeneity of pathogenic processes underlying their conditions. Ustekinumab targets the IL-12/IL-23-p40 subunit and inhibits type-1 and type-17 T-cell responses. Although ustekinumab is effective as both short- and long-term treatment, therapeutic response varies considerably among patients. Ustekinumab biosimilars will be commercialized in the very next future, likely broadening the use of this drug in the treatment of psoriasis patients. Our pharmacogenomic study evaluated the influence of 417 single-nucleotide polymorphisms (SNPs) in psoriasis-risk alleles on the clinical response to ustekinumab in a cohort of 152 patients affected by moderate-to-severe plaque-type psoriasis. Differences in SNP pattern characterizing HLA-Cw6+ or HLA-Cw6- patients, showing high or low responses to ustekinumab, were also analysed. We identified twelve SNPs in HLA-C upstream region (rs12189871, rs4406273, rs9348862 and rs9368670), PSORS1C3 (rs1265181), MICA (rs2523497), LCE3A-B intergenic region (rs12030223, rs6701730), CDSN (rs1042127, rs4713436), CCHCR1 (rs2073719) and in TNFA (rs1800610) genes associated with excellent response to ustekinumab. We also found that HLA-Cw6+ and HLA-Cw6- patients carried out distinct patterns of SNPs associated with different clinical responses. The assessment of HLA-C alleles, together with other genetic variants, could be helpful for defining patients who better benefit from anti-IL-12/IL-23 therapy.

Functional analysis of intergenic regulatory regions of genes encoding surface adhesins in Staphylococcus aureus isolates from periprosthetic joint infections

Staphylococcus aureus is a leading cause of prosthetic joint infections (PJI). Surface adhesins play an important role in the primary attachment to plasma proteins that coat the surface of prosthetic devices after implantation. Previous efforts to identify a genetic component of the bacterium that confers an enhanced capacity to cause PJI have focused on gene content, kmers, or single-nucleotide polymorphisms (SNPs) in coding sequences. Here, using a collection of S. aureus strains isolated from PJI and wounds, we investigated whether genetic variations in the regulatory region of genes encoding surface adhesins lead to differences in their expression levels and modulate the capacity of S. aureus to colonize implanted prosthetic devices. The data revealed that S. aureus isolates from the same clonal complex (CC) contain a specific pattern of SNPs in the regulatory region of genes encoding surface adhesins. As a consequence, each clonal lineage shows a specific profile of surface proteins expression. Co-infection experiments with representative isolates of the most prevalent CCs demonstrated that some lineages have a higher capacity to colonize implanted catheters in a murine infection model, which correlated with a greater ability to form a biofilm on coated surfaces with plasma proteins. Together, results indicate that differences in the expression level of surface adhesins may modulate the propensity of S. aureus strains to cause PJI. Given the high conservation of surface proteins among staphylococci, our work lays the framework for investigating how diversification at intergenic regulatory regions affects the capacity of S. aureus to colonize the surface of medical implants.

Unveiling biogeographical patterns in the worldwide distributed Ceratitis capitata (medfly) using population genomics and microbiome composition.

Invasive species are among the most important, growing threats to food security and agricultural systems. The Mediterranean medfly, Ceratitis capitata, is one of the most damaging representatives of a group of rapidly expanding species in the family Tephritidae, due to their wide host range and high invasiveness potential. Here, we used restriction site-associated DNA sequencing (RADseq) to investigate the population genomic structure and phylogeographical history of medflies collected from six sampling sites, including Africa (South Africa), the Mediterranean (Spain, Greece), Latin America (Guatemala, Brazil) and Australia. A total of 1907 single nucleotide polymorphisms (SNPs) were used to identify two genetic clusters separating native and introduced ranges, consistent with previous findings. In the introduced range, all individuals were assigned to one genetic cluster except for those in Brazil, which showed introgression of an additional genetic cluster that also appeared in South Africa, and which could not be previously identified using microsatellite markers. Moreover, we assessed the microbial composition variations in medfly populations from selected sampling sites using amplicon sequencing of the 16S ribosomal RNA (V4 region). Microbiome composition and structure were highly similar across geographical regions and host plants, and only the Brazilian specimens showed increased diversity levels and a unique composition of its microbiome compared to other sampling sites. The unique SNP patterns and microbiome features in the Brazilian specimens could point to a direct migration route from Africa with subsequent adaptation of the microbiota to the specific conditions present in Brazil. These findings significantly improve our understanding of the evolutionary history of the global medfly invasions and their adaptation to newly colonized environments.

Decomposition of Individual SNP Patterns from Mixed DNA Samples

Single-nucleotide polymorphism (SNP) markers have great potential to identify individuals, family relations, biogeographical ancestry, and phenotypic traits. In many forensic situations, DNA mixtures of a victim and an unknown suspect exist. Extracting SNP profiles from suspect’s samples can be used to assist investigation or gather intelligence. Computational tools to determine inclusion/exclusion of a known individual from a mixture exist, but no algorithm for extraction of an unknown SNP profile without a list of suspects is available. Here, we present an advanced haplotype-based HMM algorithm (AH-HA), a novel computational approach for extracting an unknown SNP profile from whole genome sequencing (WGS) of a two-person mixture. AH-HA utilizes techniques similar to the ones used in haplotype phasing. It constructs the inferred genotype as an imperfect mosaic of haplotypes from a reference panel of the target population. It outperforms more simplistic approaches, maintaining high performance through a wide range of sequencing depths (500×–5×). AH-HA can be applied in cases of victim–suspect mixtures and improves the capabilities of the investigating forces. This approach can be extended to more complex mixtures with more donors and less prior information, further motivating the development of SNP-based forensics technologies.

Characterizing Fractal Genetic Variation in the Human Genome from the Hapmap Project.

Over the last decades, the exuberant development of next-generation sequencing has revolutionized gene discovery. These technologies have boosted the mapping of single nucleotide polymorphisms (SNPs) across the human genome, providing a complex universe of heterogeneity characterizing individuals worldwide. Fractal dimension (FD) measures the degree of geometric irregularity, quantifying how "complex" a self-similar natural phenomenon is. We compared two FD algorithms, box-counting dimension (BCD) and Higuchi's fractal dimension (HFD), to characterize genome-wide patterns of SNPs extracted from the HapMap data set, which includes data from 1184 healthy subjects of eleven populations. In addition, we have used cluster and classification analysis to relate the genetic distances within chromosomes based on FD similarities to the geographical distances among the 11 global populations. We found that HFD outperformed BCD at both grand average clusterization analysis by the cophenetic correlation coefficient, in which the closest value to 1 represents the most accurate clustering solution (0.981 for the HFD and 0.956 for the BCD) and classification (79.0% accuracy, 61.7% sensitivity, and 96.4% specificity for the HFD with respect to 69.1% accuracy, 43.2% sensitivity, and 94.9% specificity for the BCD) of the 11 populations present in the HapMap data set. These results support the evidence that HFD is a reliable measure helpful in representing individual variations within all chromosomes and categorizing individuals and global populations.

An assembly-free method of phylogeny reconstruction using short-read sequences from pooled samples without barcodes

A current strategy for obtaining haplotype information from several individuals involves short-read sequencing of pooled amplicons, where fragments from each individual is identified by a unique DNA barcode. In this paper, we report a new method to recover the phylogeny of haplotypes from short-read sequences obtained using pooled amplicons from a mixture of individuals, without barcoding. The method, AFPhyloMix, accepts an alignment of the mixture of reads against a reference sequence, obtains the single-nucleotide-polymorphisms (SNP) patterns along the alignment, and constructs the phylogenetic tree according to the SNP patterns. AFPhyloMix adopts a Bayesian inference model to estimate the phylogeny of the haplotypes and their relative abundances, given that the number of haplotypes is known. In our simulations, AFPhyloMix achieved at least 80% accuracy at recovering the phylogenies and relative abundances of the constituent haplotypes, for mixtures with up to 15 haplotypes. AFPhyloMix also worked well on a real data set of kangaroo mitochondrial DNA sequences.

The development of unlabeled probes-high resolution melting (UP-HRM) marker on SAD, IAA27 and ACC genes of oil palm

Abstract. Saputra TI, Roberdi, Nugroho YA, Artutiningsih W, Purba OS, Maryanto SD, Yono D, Utomo C, Liwang T. 2021. The development of unlabeled probes-high resolution melting (UP-HRM) marker on SAD, IAA27 and ACC genes of oil palm. Biodiversitas 22: 3356-3362. The unlabeled probes-high resolution melting (UP-HRM) marker is a useful tool for detecting of single nucleotide polymorphisms (SNPs). The objectives of this study were to develop UP-HRM markers to differentiate specific SNPs patterns on oil palm. The marker was developed and tested with Elaeis guineensis (Eg), Elaeis oleifera (Eo), Eo x Eg (hybrid), and was validated with 53 individuals of BC1F1 populations ((Eo x Eg) x Eg). Four UP-HRM markers were developed based on 2 SNPs in the stearoyl-acyl-carrier-protein 9-desaturase (EgSAD), 1 SNP in the auxin-responsive protein IAA27-like (EgIAA27), and 1 SNP in the 1-amino cyclopropane-1-carboxylate oxidase (EgACC) genes. The SNP discovery result showed that Eg was represented a reference homozygote genotype, while Eo was represented as an alternative homozygote genotype and the Eo x Eg hybrid was represented as a heterozygote genotype in all genes. The typical UP-HRM melt curve graph was successfully generated. This result was consistent with each genotype model for all four markers. The UP-HRM markers can distinguish each genotype according to the single-pass sequencing results. Furthermore, dendrogram analysis on validation divided 53 BC1F1 samples into three cluster groups.

SARS-CoV-2 genomic diversity and the implications for qRT-PCR diagnostics and transmission.

The COVID-19 pandemic has sparked an urgent need to uncover the underlying biology of this devastating disease. Though RNA viruses mutate more rapidly than DNA viruses, there are a relatively small number of single nucleotide polymorphisms (SNPs) that differentiate the main SARS-CoV-2 lineages that have spread throughout the world. In this study, we investigated 129 RNA-seq data sets and 6928 consensus genomes to contrast the intra-host and inter-host diversity of SARS-CoV-2. Our analyses yielded three major observations. First, the mutational profile of SARS-CoV-2 highlights intra-host single nucleotide variant (iSNV) and SNP similarity, albeit with differences in C > U changes. Second, iSNV and SNP patterns in SARS-CoV-2 are more similar to MERS-CoV than SARS-CoV-1. Third, a significant fraction of insertions and deletions contribute to the genetic diversity of SARS-CoV-2. Altogether, our findings provide insight into SARS-CoV-2 genomic diversity, inform the design of detection tests, and highlight the potential of iSNVs for tracking the transmission of SARS-CoV-2.

HLA-Cw6 and other HLA-C alleles, as well as MICB-DT, DDX58, and TYK2 genetic variants associate with optimal response to anti-IL-17A treatment in patients with psoriasis

ABSTRACT Objective: Our pharmacogenomic study evaluated the influence of the presence/absence of genetic variants of psoriasis-risk loci on the clinical response to secukinumab. Differences in the single-nucleotide polymorphism (SNP) pattern characterizing HLA-Cw6+ or HLA-Cw6− patient subpopulations, showing high or low responses to secukinumab, were also analyzed. Methods: 417 SNPs were analyzed by Next-Generation Sequencing technology, in a cohort of 62 psoriatic patients and undergone secukinumab treatment. Univariate regression analysis was employed to examine the association between SNP and clinical response to secukinumab. Multivariate analysis was also performed to assess multivariate differences in SNP pattern of HLA-Cw6+ or HLA-Cw6− patients showing high or low responses to secukinumab. Results: Eight SNPs in HLA-C and upstream region (rs13207315, rs6900444, rs12189871, rs12191877, rs4406273, and rs10484554), including HLA-Cw6 classical allele (rs1131118), and three in MICB-DT (rs9267325), DDX58 (rs34085293) and TYK2 (rs2304255) genes, associating with excellent response to secukinumab were identified. Importantly, rs34085293 or rs2304255 SNP status defined a subgroup of super-responder patients. We also found that HLA-Cw6+ and HLA-Cw6− patients carried out specific patterns of SNPs associating with different responses to secukinumab. Conclusion: Assessment of HLA-Cw6, together with other allelic variants of genes, could be helpful to define patients which better benefit from anti-IL-17 therapy. Abbreviations: PASI: Psoriasis Area and Severity Index; SNP: Single-Nucleotide Polymorphism Rs: Reference SNP; PASI75: 75% reduction in Psoriasis Area and Severity Index; PASI90: 90% reduction in Psoriasis Area and Severity Index; PASI100: 100% reduction in Psoriasis Area and Severity Index; NGS: Next-Generation Sequencing; OR: Odds Ratio; CAP: Canonical Analysis of Principal coordinates; BMI: Body Mass Index; LD: Linkage Disequilibrium

Mitochondrial DNA Repair in an Arabidopsis thaliana Uracil N-Glycosylase Mutant.

Substitution rates in plant mitochondrial genes are extremely low, indicating strong selective pressure as well as efficient repair. Plant mitochondria possess base excision repair pathways; however, many repair pathways such as nucleotide excision repair and mismatch repair appear to be absent. In the absence of these pathways, many DNA lesions must be repaired by a different mechanism. To test the hypothesis that double-strand break repair (DSBR) is that mechanism, we maintained independent self-crossing lineages of plants deficient in uracil-N-glycosylase (UNG) for 11 generations to determine the repair outcomes when that pathway is missing. Surprisingly, no single nucleotide polymorphisms (SNPs) were fixed in any line in generation 11. The pattern of heteroplasmic SNPs was also unaltered through 11 generations. When the rate of cytosine deamination was increased by mitochondrial expression of the cytosine deaminase APOBEC3G, there was an increase in heteroplasmic SNPs but only in mature leaves. Clearly, DNA maintenance in reproductive meristem mitochondria is very effective in the absence of UNG while mitochondrial genomes in differentiated tissue are maintained through a different mechanism or not at all. Several genes involved in DSBR are upregulated in the absence of UNG, indicating that double-strand break repair is a general system of repair in plant mitochondria. It is important to note that the developmental stage of tissues is critically important for these types of experiments.

Mitochondrial D-loop informative SNPs in identification of dog’s breed

The aim of the study was to determine differences at the molecular level between different breeds of dogs on the basis of a comparative analysis of the mtDNA D-loop sequence. The material used in the study consisted of blood collected from 40 Doberman Pinscher, Dachshund, German Shepherd, and Crossbreed dogs. The investigations involved DNA extraction, amplification and sequencing of the mtDNA D-loop, as well as bioinformatic analyses. Fifteen SNP (single nucleotide polymorphism) mutations were identified. This study proposes a new solution for dog breed identification, i.e. the use of SNPs specific to each breed. The analysis revealed the presence of already known SNPs in various configurations depending on the breed. Breed-specific SNP patterns were identified in 94% of the Doberman dogs, and two SNP patterns were found in 73% of the German Shepherd dogs. This subject requires further research. However, given the limited amount of data on the use of mtDNA in determination of breed affiliation, the present results seem to be promising.

SNP and indel frequencies at transcription start sites and at canonical and alternative translation initiation sites in the human genome.

Single-nucleotide polymorphisms (SNPs) are the most common form of genetic variation in humans and drive phenotypic variation. Due to evolutionary conservation, SNPs and indels (insertion and deletions) are depleted in functionally important sequence elements. Recently, population-scale sequencing efforts such as the 1000 Genomes Project and the Genome of the Netherlands Project have catalogued large numbers of sequence variants. Here, we present a systematic analysis of the polymorphisms reported by these two projects in different coding and non-coding genomic elements of the human genome (intergenic regions, CpG islands, promoters, 5’ UTRs, coding exons, 3’ UTRs, introns, and intragenic regions). Furthermore, we were especially interested in the distribution of SNPs and indels in direct vicinity to the transcription start site (TSS) and translation start site (CSS). Thereby, we discovered an enrichment of dinucleotides CpG and CpA and an accumulation of SNPs at base position −1 relative to the TSS that involved primarily CpG and CpA dinucleotides. Genes having a CpG dinucleotide at TSS position -1 were enriched in the functional GO terms “Phosphoprotein”, “Alternative splicing”, and “Protein binding”. Focusing on the CSS, we compared SNP patterns in the flanking regions of canonical and alternative AUG and near-cognate start sites where we considered alternative starts previously identified by experimental ribosome profiling. We observed similar conservation patterns of canonical and alternative translation start sites, which underlines the importance of alternative translation mechanisms for cellular function.

Deletion of biosynthetic genes, specific SNP patterns and differences in transcript accumulation cause variation in hydroxynitrile glucoside content in barley cultivars

Barley (Hordeum vulgare L.) produces five leucine-derived hydroxynitrile glucosides, potentially involved in alleviating pathogen and environmental stresses. These compounds include the cyanogenic glucoside epiheterodendrin. The biosynthetic genes are clustered. Total hydroxynitrile glucoside contents were previously shown to vary from zero to more than 10,000 nmoles g−1 in different barley lines. To elucidate the cause of this variation, the biosynthetic genes from the high-level producer cv. Mentor, the medium-level producer cv. Pallas, and the zero-level producer cv. Emir were investigated. In cv. Emir, a major deletion in the genome spanning most of the hydroxynitrile glucoside biosynthetic gene cluster was identified and explains the complete absence of hydroxynitrile glucosides in this cultivar. The transcript levels of the biosynthetic genes were significantly higher in the high-level producer cv. Mentor compared to the medium-level producer cv. Pallas, indicating transcriptional regulation as a contributor to the variation in hydroxynitrile glucoside levels. A correlation between distinct single nucleotide polymorphism (SNP) patterns in the biosynthetic gene cluster and the hydroxynitrile glucoside levels in 227 barley lines was identified. It is remarkable that in spite of the demonstrated presence of a multitude of SNPs and differences in transcript levels, the ratio between the five hydroxynitrile glucosides is maintained across all the analysed barley lines. This implies the involvement of a stably assembled multienzyme complex.