Oligonucleotide Primers Research Articles

Application of a new highly multiplexed amplicon sequencing tool to evaluate Plasmodium falciparum antimalarial resistance and relatedness in individual and pooled samples from Dschang, Cameroon.

Resistance to antimalarial drugs remains a major obstacle to malaria elimination. Multiplexed, targeted amplicon sequencing is being adopted for surveilling resistance and dissecting the genetics of complex malaria infections. Moreover, genotyping of parasites and detection of molecular markers drug resistance in resource-limited regions requires open-source protocols for processing samples, using accessible reagents, and rapid methods for processing numerous samples including pooled sequencing. P lasmodium falciparum S treamlined M ultiplex A ntimalarial R esistance and R elatedness T esting ( Pf -SMARRT) is a PCR-based amplicon panel consisting of 15 amplicons targeting antimalarial resistance mutations and 9 amplicons targeting hypervariable regions. This assay uses oligonucleotide primers in two pools and a non-proprietary library and barcoding approach. We evaluated Pf -SMARRT using control mocked dried blood spots (DBS) at varying levels of parasitemia and a mixture of 3D7 and Dd2 strains at known frequencies, showing the ability to genotype at low parasite density and recall within-sample allele frequencies. We then piloted Pf -SMARRT to genotype 100 parasite isolates collected from uncomplicated malaria cases at three health facilities in Dschang, Western Cameroon. Antimalarial resistance genotyping showed high levels of sulfadoxine-pyrimethamine resistance mutations, including 31% prevalence of the DHPS A613S mutation. No K13 candidate or validated artemisinin partial resistance mutations were detected, but one low-level non-synonymous change was observed. Pf -SMARRT's hypervariable targets, used to assess complexity of infections and parasite diversity and relatedness, showed similar levels and patterns compared to molecular inversion probe (MIP) sequencing. While there was strong concordance of antimalarial resistance mutations between individual samples and pools, low-frequency variants in the pooled samples were often missed. Overall, Pf -SMARRT is a robust tool for assessing parasite relatedness and antimalarial drug resistance markers from both individual and pooled samples. Control samples support that accurate genotyping as low as 1 parasite per microliter is routinely possible. Malaria remains a critical global public health problem. Antimalarial drug resistance has repeatedly undermined control and the emergence of artemisinin partial resistance in Africa is the latest major challenge. Malaria molecular surveillance (MMS) has emerged as a powerful tool to monitor molecular markers of resistance and changes in the parasite population. Streamlined methods are needed that can be readily adopted in endemic countries. We developed P lasmodium falciparum S treamlined M ultiplex A ntimalarial R esistance and R elatedness T esting ( Pf -SMARRT), a multiplex amplicon deep sequencing approach that uses easily accessible products without proprietary steps and can be sequenced on any Illumina sequencer. We validated this tool using controls, including mocked dried blood spots, and then implemented it to evaluate resistance and parasite relatedness among 100 samples from Cameroon. The assay was able to reliably assess the within-sample allele frequency of antimalarial resistance markers and discriminate strains within and between individuals. We also evaluated a more cost-effective surveillance approach for antimalarial resistance polymorphisms using pooled samples. While within-pool frequencies of mutations were accurate in pools with higher numbers of samples, this resulted in the loss of the ability to detect variants uncommon in the pool. Overall Pf- SMARRT provides a new protocol for conducting MMS that is easily implementable in Africa.

Unraveling the abundance of vip3-type genes in Indian Bacillus thuringiensis across the agroclimatic landscape and impact of amino acid substitutions for safer agriculture

Vegetative insecticidal protein (vip) genes of Bacillus thuringiensis (Bt) are candidates for gene pyramiding in the resistance management of pests. The prevalence of vip genes in Bt isolates is relatively under-explored. Bt isolates recovered from 29 diverse sources in nine agro-climatic zones of India were screened for the presence of vip3-type genes by PCR with 4 sets of oligonucleotide primers. Out of 155 Bt isolates, 70.32 % (109) and 44.52 % (69) isolates were positive for amplification of partial vip3-type genes with primer sets 1 and 4, respectively. The primer set-2 was found to be more efficient for amplifying full-length genes (29.03 % /45 isolates) as compared with primer set-3 (3.23 %/ 5 isolates), also corroborated in the amplification of full-length vip3 genes in ten Bt BGSC strains used as reference. Frequency analysis revealed presence of vip3 genes in Bt isolates across all agro-climatic zones. Thus, Indian Bt isolates from diverse sources have a rich repertoire of vip3-type genes. Our study reports the highest number (45) of full-length vip3-type genes detected in a native Bt isolates collection, demonstrating enrichment of Indian Bt isolates for vip3 genes. Twelve of these genes have been cloned, sequenced, and out of these, six were found to be effective against Helicoverpa armigera in our laboratory previously. Comparison of substitutions in deduced amino acids sequence of these genes and expression of Vip3 proteins in SDS-PAGE analysis of selected native Bt isolates positive for full-length vip3-type genes indicated their biopesticidal potential.

Molecular identification of Baylisascaris melis (Gedoelst, 1920) from the Eurasian badger (Meles meles) and ascarids from other wild carnivores in Kazakhstan.

The presence of gastrointestinal nematodes, including zoonotic ascarids, in wild canids, felids and mustelids as definitive hosts in Central Asian countries has been documented in many studies based on traditional morphological methods. In contrast, relevant data for the badger are scarce. The aim of this study was the molecular identification of ascarid nematodes from five wild carnivore species in different regions of Kazakhstan. A total of 211 adult ascarids were collected from gray wolves (Canis lupus, 8 of 83 infected with 2-6 Toxascaris leonina), red foxes (Vulpes vulpes, 26 of 53, with 2-8 Toxascaris leonina), corsac foxes (Vulpes corsac, 6 of 11, 3-6 Toxascaris leonina), lynx (Lynx lynx, 2 of 3, with 2-5 Toxocara cati) and badgers (Meles meles, 2 of 4, with 2-7 Baylisascaris melis). Genomic DNA was extracted from the worms and ribosomal DNA, including the first and second internal transcribed spacer genes, was amplified by polymerase chain reaction using specific oligonucleotide primers and then sequenced. Toxascaris leonina, but not Toxocara canis, was molecularly identified in the wild canids, Toxocara cati in the lynx and Baylisascaris melis in the badger. The maximum likelihood phylogenetic tree showed three distinct clades: the canid Toxascaris leonina was placed in one clade, Toxocara cati in another and Baylisascaris melis in a third. The study provides the world's first molecular data and phylogenetic analysis of Baylisascaris melis, identified for the second time since its description over 100 years ago. This species was shown to be genetically distinct from other Baylisascaris spp. (B. columnaris, B. procyonis, B. transfuga, B. devosi). The possible zoonotic significance of ascarids from wild carnivores is discussed in the light of conditions in Central Asia.

Cloning and expression of UbiA human gene using innovative methodologies for recombinant protein production in PUAST vector

Introduction: A systematic library of Gal4/UAS-regulated transgenes has proven to be a powerful genetic system for identifying genes and defining pathways in development. This system offers valuable insights that highlight the evolutionary conservation between animals and humans. Objectives: The objective of this study was to clone, express, and characterize the UbiA gene. The research presents a highly effective method for cloning genes, using the UbiA-pcDNA3 gene as a model for mammalian cloning. These genes were then integrated into the PUAST vector of Drosophila, an expression vector and eukaryotic cell system commonly used for producing recombinant proteins. Materials and Methods: UbiA was isolated from human cells, and complementary DNA was synthesized. An oligonucleotide primer pair was designed based on the UbiA gene sequence, incorporating XhoI and Xbal restriction sites at the 5´ end of the forward and reverse primers, respectively. The UbiA gene was then amplified by PCR, cloned into the pcDNA3 plasmid, and the resulting recombinant plasmid was sequenced. Subsequently, the gene was sub cloned into the PUAST vector and expressed in S2 cells as a eukaryotic cell system. Protein determination and verification were conducted through western blotting techniques. Results: Confirmation of UbiA gene cloning into the PUAST vector was achieved through colony-PCR and digestion by enzymes. Cloning and sub cloning techniques validated by enzymatic digestion, along with gene sequencing. The identity between cloned UbiA gene and the identical gene exhibited 99%. We revealed a singular band purified protein through western blotting with 60 kDa size. Conclusion: More protein synthesis of the UbiA gene can be achieved by using the eukaryotic expression system provided by the PUAST vector. This technique has been proven to be a suitable platform and can be instrumental in various applications such as therapeutics, pharmacology, and vaccine development.

Association of Telomere Length in T Lymphocytes, B Lymphocytes, NK Cells and Monocytes with Different Forms of Age-Related Macular Degeneration.

Age plays a primary role in the development of age-related macular degeneration (AMD). Telomere length (TL) is one of the most relevant biomarkers of aging. In our study, we aimed to determine the association of TL with T lymphocytes, B lymphocytes, NK cells or monocytes with different forms of AMD. Our study included 62 patients with AMD: geographic atrophy (GA), neovascular AMD (NVAMD) with and without macular atrophy and 22 healthy controls. Each leukocyte subtype was isolated from peripheral blood by immunomagnetic separation, and the DNA was purified. The TL in the genomic DNA was determined using qPCR by amplifying the telomere region with specific oligonucleotide primers and normalizing to the control gene. Statistical analysis was performed using R version 4.5.1. We observed a statistically significant increase in TL in the T cells between the control and NVAMD groups but not for the GA group. The B cells and monocytes showed a significant decrease in TL in all AMD groups. The TL in the NK cells did not decrease in any of the AMD groups. The TL in the monocytes had the strongest association with AMD. It reflects a person's "telomeric status" and may become a diagnostic hallmark of these degenerative processes.

ECPUB5 Polyubiquitin Gene in Euphorbia characias: Molecular Characterization and Seasonal Expression Analysis.

The spurge Euphorbia characias is known for its latex, which is rich in antioxidant enzymes and anti-phytopathogen molecules. In this study, we identified a novel polyubiquitin protein in the latex and leaves, leading to the first molecular characterization of its coding gene and expressed protein in E. characias. Using consensus-degenerate hybrid oligonucleotide primers (CODEHOP) and rapid amplification of cDNA ends (5'/3'-RACE), we reconstructed the entire open reading frame (ORF) and noncoding regions. Our analysis revealed that the polyubiquitin gene encodes five tandemly repeated sequences, each coding for a ubiquitin monomer with amino acid variations in four of the five repeats. In silico studies have suggested functional differences among monomers. Gene expression peaked during the summer, correlating with high temperatures and suggesting a role in heat stress response. Western blotting confirmed the presence of polyubiquitin in the latex and leaf tissues, indicating active ubiquitination processes. These findings enhance our understanding of polyubiquitin's regulatory mechanisms and functions in E. characias, highlighting its unique structural and functional features.

Assessment of Genetic Diversity in Tomato (Solanum lycopersicum) Varieties by Using RAPD Markers

Aim: Assessment of genetic diversity in Tomato (Solanum lycopersicum) varieties by using RAPD markers. Place and Duration of Study: Department of Plant Biotechnology of K. K. Wagh College of Agricultural Biotechnology, Nashik, Maharashtra. Methodology: Tomato is one of the most important vegetable crops in the world and normally human being daily consumed in diet. The five Varities were collected from Mahalaxmi Hi-Tech Nursery Nashik. The present study was conducted at K K Wagh college of Agricultural Biotechnology Nashik. DNA was isolated by using modified CTAB method Five Varities were analyzed through RAPD markers to determine the molecular characterization at individual level. Results: Six arbitrary oligonucleotide primers used in the RAPD-PCR produced a total 38 different marker bands with an average 8 band per primer. Based on the banding pattern 100% polymorphism observed among the tomato varieties. Size range of amplified DNA bands varied from 0.1-10 kb. A total of 14 unique bands were amplified from the genome of the 5 tomato varieties. The dendrogram clustered the tomato varieties into two major clusters. The clusters were subdivided into small groups including the highly related Keshar and 0225, Anisha and Sai-22 varieties. The other Cluster included an isolated variety of Aryaman as a distinctive one. The RAPD data revealed a high genetic polymorphism that can be used to select diverse parents in breeding programs and maintain genetic variation.

Broad-range amplification and sequencing of the rpoB gene: a novel assay for bacterial identification in clinical microbiology.

The rpoB gene has been proposed as a promising phylogenetic marker for bacterial identification, providing theoretically improved species-level resolution compared to the 16S rRNA gene for a range of clinically important taxa. However, its utility in diagnostic microbiology has been limited by the lack of broad-range primers allowing for its amplification from most species with a single PCR assay. Here, we present an assay for broad-range partial amplification and Sanger sequencing of the rpoB gene. To reduce cross-reactivity and allow for rpoB amplification directly from patient samples, primers were based on the dual priming oligonucleotide principle. The resulting amplicon is ~550 base pairs in length and appropriate for species-level identification. Systematic in silico evaluation of a wide selection of taxa demonstrated improved resolution within multiple important genera, including Enterococcus, Fusobacterium, Mycobacterium, Streptococcus, and Staphylococcus species and several genera within the Enterobacteriaceae family. Broad-range rpoB amplification and Sanger sequencing of 115 bacterial isolates provided unambiguous species-level identification for 97 (84%) isolates, as compared to 57 (50%) using a clinical 16S rRNA gene assay. Several unresolved taxonomic matters disguised by the low resolution of the 16S rRNA gene were revealed using the rpoB gene. Using a collection of 33 clinical specimens harboring bacteria and assumed to contain high concentrations of human DNA, the rpoB assay identified the pathogen in 29 specimens (88%). Broad-range rpoB amplification and sequencing provides a promising tool for bacterial identification, improving discrimination between closely related species and making it amenable for use in culture-based and culture-independent diagnostic approaches.

Single-round QuikChange PCR for engineering multiple site-directed mutations in plasmid DNA

Mutational study is a cornerstone methodology in biochemistry and genetics, and many mutagenesis strategies have been invented to promote the efficiency of gene engineering. In this study, we developed a simple and timesaving approach to integrate simultaneous mutagenesis at discrete sites. By using plasmid as a template and compatible oligonucleotide primers per the QuikChange strategy, our method was able to introduce multiple nucleotide insertions, deletions and replacements in one round of polymerase chain reaction. The longest insertion and deletion were achieved with 28 bp and 16 bp mismatch respectively. For minor nucleotide replacements (mismatch no more than 4 bp), mutations were achieved at up to 4 discrete locations. Usually, a successful clone with all desired mutations was found by screening 5 colonies. Clones with a subset of mutations may be stocked into the library of mutants or used as templates in the next rounds of mutagenic PCR to accomplish the entire construction project. This method can be applied to build up a combinatory library of mutants through saturation mutagenesis at multiple sites. It is promising to facilitate the research of protein biochemistry, forward genetics and synthetic biology.

Development of rapid and efficient enterovirus typing and classification platform for real-time diagnostics

Enteroviruses are highly prevalent among children and adults worldwide. Accurate detection and genotyping of enteroviruses can be achieved using consensus-degenerate hybrid oligonucleotide primer polymerase chain reaction (PCR)-based methods. Nevertheless, the popularized use of such methods for enterovirus detection and genotyping is limited by their relatively long processing time (>2 days), high processing cost, and the requirement for specialized and expensive instrumentation. Here, we report a relatively simple asymmetric PCR-electrochemical biosensing method that can detect enteroviruses and accurately characterize them into four distinct genotypic subgroups in less than 2 h. Specifically, genes of interest in the enteroviruses were amplified via asymmetric PCR (asy-PCR) using the designed PCR primers. The asy-PCR products were hybridized with enterovirus genotype-specific single-stranded DNA primer probes. Changes in response to electrochemical impedance signals can rapidly clarify the genotypes of enteroviruses. We observed that this method could not only effectively amplify the target gene within minimal PCR-amplified cycles of consensus-degenerate hybrid oligonucleotide (COHOP) products from the specimen but also allowed the rapid differentiation of the four subgroups of enteroviruses in 2 h. In summary, we developed an efficient and rapid electrochemical biosensing platform for enterovirus subtyping diagnostics that can be applied to other emergent infectious diseases.

A novel digital PCR assay for detection and comprehensive characterization of Molluscum contagiosum virus genotypes MOCV1, MOCV2, and MOCV3 and recombinant lineages

Molluscum contagiosum virus (MOCV) is an important human pathogen causing a high disease burden worldwide. It is the last exclusively human-infecting poxvirus still circulating in its natural reservoir—a valuable model of poxviral evolution. Unfortunately, MOCV remains neglected, and little is known about its evolutionary history and circulating genomic variants, especially in non-privileged countries. The design weaknesses of available MOCV detection/genotyping assays surfaced with recent accumulation of abundant sequence information: all existing MOCV assays fail at accurate genotyping and capturing sub-genotype level diversity. Because complete MOCV genome characterization is an expensive and labor-intensive task, it makes sense to prioritize samples for whole-genome sequencing by diversity triage screening. To meet this demand, we developed a novel assay for accurate MOCV detection and genotyping, and comprehensive sub-genotype qualification to the level of phylogenetic groups (PGs). The assay included a novel set of oligonucleotide primers and probes, and it was implemented using digital polymerase chain reaction (dPCR). It offers sensitive, specific, and accurate detection, genotyping (MOCV1–MOCV3), and PG qualification (PG1–6) of MOCV DNA from clinical samples. The novel dPCR assay is suitable for MOCV diversity triage screening and prioritization of samples for complete MOCV genome characterization.

Detection fimH Gene in The Urine of UTI Patients at The Puskesmas Banyuanyar, as a Marker for The Presence of Escherichia coli

Urinary Tract Infection (UTI) is a bacterial infection characterized by a urine culture showing bacteriuria with bacterial counts exceeding 100,000 per milliliter of urine. Polymerase Chain Reaction (PCR) is a method that utilizes in-vitro enzymatic reactions to amplify specific DNA targets. This process synthesizes new DNA strands complementary to the target sequences using enzymes and oligonucleotide primers within a thermocycler. The fimH gene is among the most important of the UPEC strains and is associated with severe urinary tract infections. The aim of this study was to identify the presence of the fimH gene in urine samples from UTI patients at the Puskesmas Banyuanyar in Surakarta using Polymerase Chain Reaction (PCR). The research employed the Quota Sampling Technique, collecting a total of five urine samples from individuals diagnosed with urinary tract infections. The results of the control isolates using pure Escherichia coli isolates showed that the fimH gene PCR amplified control with annealing temperature of 52°C. The analysis of clinical isolates of Escherichia coli from the urine of urinary tract infection patients revealed that the fimH gene was PCR amplified in all five samples.

Validation of Single-Nucleotide Mosaic Variants Through Droplet Digital PCR.

The detection, validation, and subsequent interpretation of potentially mosaic single-nucleotide variants (SNV) within next-generation sequencing data remains a challenge in both research and clinical laboratory settings. The ability to identify mosaic variants in high genome coverage sequencing data at levels of ≤1% underscores the necessity for developing guidelines and best practices to verify these variants orthogonally. Droplet digital PCR (ddPCR) has proven to be a powerful and precise method that allows for the determination of low-level variant fractions within a given sample. Herein we describe two precise ddPCR methods using either a fluorescent TaqMan hydrolysis probe approach or an EvaGreen fluorescent dye protocol. The TaqMan approach relies on two different fluorescent probes (FAM and HEX/VIC), each designed to amplify selectively only in the presence of a single nucleotide change denoting the variant or reference position. The fractional abundance is then calculated to determine the relative quantities of both alleles in the final sample. The EvaGreen protocol relies on two independent reactions with oligonucleotide primers designed with the single nucleotide change denoting the variant at the penultimate position of the primer. The relative amplification efficiency of both primer sets (reference and variant) can be compared to determine the mosaic level of a given variant. As the cost of high-coverage sequencing continues to decrease, the identification of potentially mosaic variants will also increase. The approaches outlined will allow clinicians and researchers a more precise determination of the true mosaic level of a given variant allowing them to better assess not only its potential pathogenicity but also its possible recurrence risk when offering genetic counseling to families. © 2024 Wiley Periodicals LLC. Basic Protocol: Droplet digital PCR (ddPCR) with TaqMan hydrolysis probes Alternate Protocol: EvaGreen oligonucleotide-specific ddPCR.

Assessment of Torque Teno Virus (TTV) Frequency in Healthy Blood Donors in the Central Region of Iran, Yazd.

Background: Torque teno virus (TTV) is a globally prevalent virus in humans, yet comprehensive knowledge about its prevalence, predominant transmission routes, and pathogenesis remains limited. This study aimed to assess the frequency of TTV infection among healthy blood donors in Yazd, Iran. Materials and Methods: A total of 236 healthy blood donors, devoid of HIV/HBV/HCV infection markers, participated in the study from 2015 to 2016. Nested Polymerase Chain Reaction (PCR) utilizing a set of oligo primers for the 5΄- UTR region was employed to detect TTV DNA in serum samples. Results: The TTV genome was identified in 161 out of 236 (61.2%) healthy blood donors. The mean age for men and women was 43 and 57 years, respectively. Of the participants, 156 were male, and 107 were female. Donor age exhibited a significant association with virus presence (P=0.007); however, gender did not show a statistically significant association with the frequency of TTV infection in healthy blood donors (P=0.3). Conclusion: The study revealed a notably high frequency of the Torque teno virus in Yazd province, aligning with similar findings globally. Further investigations are warranted to elucidate the clinical implications of the virus in the healthy population.

Aberrant methylation of placental development genes in chorionic villi of spontaneous abortions with trisomy 16.

In humans, aneuploidy is incompatible with the birth of healthy children and mainly leads to the death of embryos in the early stages of development in the first trimester of pregnancy. Trisomy 16 is the most common aneuploidy among spontaneous abortions of the first trimester of pregnancy. However, the mechanisms leading to the death of embryos with trisomy 16 remain insufficiently investigated. One of these potential mechanisms is abnormal placental development, including aberrant remodeling of spiral arteries. Spiral artery remodeling involves the migration of trophoblast cells into the maternal spiral arteries, replacing their endothelium and remodeling to ensure a stable embryonic nutrition and oxygen supply. This is a complex process which depends on many factors from both the embryo and the mother. We analyzed the methylation level of seven genes (ADORA2B, NPR3, PRDM1, PSG2, PHTLH, SV2C, and TICAM2) involved in placental development in the chorionic villi of spontaneous abortions with trisomy 16 (n = 14), compared with spontaneous abortions with a normal karyotype (n = 31) and the control group of induced abortions (n = 10). To obtain sequencing libraries, targeted amplification of individual gene regions using designed oligonucleotide primers for bisulfite-converted DNA was used. The analysis was carried out using targeted bisulfite massive parallel sequencing. In the group of spontaneous abortions with trisomy 16, the level of methylation of the PRDM1 and PSG2 genes was significantly increased compared to induced abortions (p = 0.0004 and p = 0.0015, respectively). In the group of spontaneous abortions, there was no increase in the level of methylation of the PRDM1 and PSG2 genes, but the level of methylation of the ADORA2B gene was significantly increased compared to the induced abortions (p = 0.032). The results obtained indicate the potential mechanisms of the pathogenetic effect of trisomy 16 on the placental development with the participation of the studied genes.

Detection of Clarithromycin Resistance in Helicobacter pylori Using MmaxSure™ H. pylori & ClaR Assay

Introduction: Clarithromycin resistance is a crucial factor in the eradication of Helicobacter pylori. This study aimed to evaluate the performance of MmaxSure™ H. pylori & ClaR Assay (MmaxSure™) in the diagnosis and detection of clarithromycin resistance in H. pylori. Methods: Subjects who underwent esophagogastroduodenoscopy between April 2020 and October 2022 were enrolled. The diagnostic performances of MmaxSure™ and dual priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) were compared with the rapid urease test and culture. Secondary gene sequencing analysis was performed in discordant cases of PCR tests. Results: A total of 156 gastric biopsy samples were analyzed. In H. pylori detection, MmaxSure™ showed a 95.9% sensitivity (95% CI: 90.6–98.6), a 42.7% specificity (95% CI: 26.3–60.7), and a kappa value of 0.457. For the detection of A2143G mutation samples, MmaxSure™ showed a 91.2% sensitivity (95% CI: 76.3–98.1), a 93.4% specificity (95% CI: 87.5–97.1), and a kappa value of 0.804. There were a total of 10 discordant cases compared to gene sequencing in A2143G mutation detection for MmaxSure™. Conclusion: In this study, MmaxSure™ showed comparable diagnostic performance to DPO-PCR in the detection of the H. pylori and A2143G mutation. Further research is needed to confirm the clinical effectiveness of the MmaxSure™ assay in H. pylori eradication.

The Soybean Rpp3 Gene Encodes a TIR-NBS-LRR Protein that Confers Resistance to Phakopsora pachyrhizi.

Soybean rust is an economically significant disease caused by the fungus Phakopsora pachyrhizi that negatively impacts soybean (Glycine max [L.] Merr.) production throughout the world. Susceptible plants infected by P. pachyrhizi develop tan-colored lesions on the leaf surface that give rise to funnel-shaped uredinia as the disease progresses. While most soybean germplasm is susceptible, seven genetic loci (Rpp1 to Rpp7) that provide race-specific resistance to P. pachyrhizi (Rpp) have been identified. Rpp3 was first discovered and characterized in the soybean accession PI 462312 (Ankur), and it was also determined to be one of two Rpp genes present in PI 506764 (Hyuuga). Genetic crosses with PI 506764 were later used to fine-map the Rpp3 locus to a 371-kb region on chromosome 6. The corresponding region in the susceptible Williams 82 (Wm82) reference genome contains several homologous nucleotide binding site-leucine rich repeat (NBS-LRR) genes. To identify Rpp3, we designed oligonucleotide primers to amplify Rpp3 candidate (Rpp3C) NBS-LRR genes at this locus from PI 462312, PI 506764, and Wm82 using polymerase chain reaction (PCR). Five Rpp3C genes were identified in both Rpp3-resistant soybean lines, and co-silencing these genes compromised resistance to P. pachyrhizi. Gene expression analysis and sequence comparisons of the Rpp3C genes in PI 462312 and PI 506764 suggest that a single candidate gene, Rpp3C3, is responsible for Rpp3-mediated resistance. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Degenerate oligonucleotide primer MIG-seq: an effective PCR-based method for high-throughput genotyping.

Next-generation sequencing (NGS) library construction often involves using restriction enzymes to decrease genome complexity, enabling versatile polymorphism detection in plants. However, plant leaves frequently contain impurities, such as polyphenols, necessitating DNA purification before enzymatic reactions. To overcome this problem, we developed a PCR-based method for expeditious NGS library preparation, offering flexibility in number of detected polymorphisms. By substituting a segment of the simple sequence repeat sequence in the MIG-seq primer set (MIG-seq being a PCR method enabling library construction with low-quality DNA) with degenerate oligonucleotides, we introduced variability in detectable polymorphisms across various crops. This innovation, named degenerate oligonucleotide primer MIG-seq (dpMIG-seq), enabled a streamlined protocol for constructing dpMIG-seq libraries from unpurified DNA, which was implemented stably in several crop species, including fruit trees. Furthermore, dpMIG-seq facilitated efficient lineage selection in wheat and enabled linkage map construction and quantitative trait loci analysis in tomato, rice, and soybean without necessitating DNA concentration adjustments. These findings underscore the potential of the dpMIG-seq protocol for advancing genetic analyses across diverse plant species.

A single-cell genomic strategy for alternative transcript start sites identification.

Alternative transcription start sites (TSSs) usage plays a critical role in gene transcription regulation in mammals. However, precisely identifying alternative TSSs remains challenging at the genome-wide level. We report a single-cell genomic technology for alternative TSSs annotation and cell heterogeneity detection. In the method, we utilize Fluidigm C1 system to capture individual cells of interest, SMARTer cDNA synthesis kit to recover full-length cDNAs, then dual priming oligonucleotide system to specifically enrich TSSs for genomic analysis. We apply this method to a genome-wide study of alternative TSSs identification in two different IFN-β stimulated mouse embryonic fibroblasts (MEFs). The data clearly discriminate two IFN-β stimulated MEFs. Moreover, our results indicate 81% expressed genes in these two cell types containing multiple TSSs, which is much higher than previous predictions based on Cap-Analysis Gene Expression (CAGE) (58%) or empirical determination (54%) in various cell types. This indicates that alternative TSSs are more pervasive than expected and implies our strategy could position them at an unprecedented sensitivity. It would be helpful for elucidating their biological insights in future.

Probing the legitimate initiation of RNA synthesis by Qβ replicase with oligonucleotide primers.

Oligoribonucleotides complementary to the template 3' terminus were tested for their ability to initiate RNA synthesis on legitimate templates capable of exponential amplification by Qβ replicase. Oligonucleotides shorter than the distance to the nearest predicted template hairpin proved able to serve as primers, with the optimal length varying for different templates, suggesting that during initiation the template retains its native fold incorporating the 3' terminus. The priming activity of an oligonucleotide is greatly enhanced by its 5'-triphosphate group, the effect being strongly dependent on Mg2+ ions. This indicates that, unlike other studied RNA polymerases, Qβ replicase binds the 5'-triphosphate of the initiating nucleotide GTP, and this binding is needed for the replication of legitimate templates.