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The tetra-primer amplification refractory mutation system–polymerase chain (ARMS–PCR) reaction is a simple and economical method to genotype single-nucleotide polymorphisms (SNPs). It uses four primers in a single PCR and is followed just by gel electrophoresis. However, the optimization step can be very hardworking and time-consuming. Hence, we propose to demonstrate and discuss critical steps for its development, in a way to provide useful information. Two SNPs that provided different amplification conditions were selected. DNA extraction methods, annealing temperatures, PCR cycles protocols, reagents, and primers concentration were also analyzed. The use of tetra-primer ARMS–PCR could be impaired for SNPs in DNA regions rich in cytosine and guanine and for samples with DNA not purified. The melting temperature was considered the factor of greater interference. However, small changes in the reagents concentration significantly affect the PCR, especially MgCl₂. Balancing the inner primers band is also a key step. So, in order to balance the inner primers band, intensity is important to observe which one has the weakest band and promote its band by increasing its concentration. The use of tetra-primer ARMS–PCR attends the expectations of modern genomic research and allows the study of SNPs in a fast, reliable, and low-cost way.

Genetics plays a crucial role in determining individual susceptibility to dental caries. Certain ( ) gene variants may be linked to oral diseases. One of the most prevalent problems with oral health is dental caries. Between 60% and 90% of schoolchildren and the vast majority of adults suffer from dental caries. This study aimed to examine the genetic association between the gene variants rs11362 and rs1799946 and dental caries in a case-control study. The study included 202 participants, of whom 119 demonstrated high dental caries, while 83 acted as controls with low dental caries. Most of them were referred to the Medical University's Dental Clinic in Shiraz, Iran. Following the acquisition of written and informed consent, genomic DNA was extracted from peripheral blood. Tetra-primer ARMS-PCR was employed to identify the promoter region polymorphisms rs11362 and rs1799946. Allelic, genotypic, and haplotypes frequencies did not differ between the case and control groups, according to the genotyping results. To find the dental caries-vulnerable loci in our case and control groups, more studies with a bigger sample size are essential. Furthermore, functional research is necessary to elucidate how these variations affect expression.

Sugarcane (Saccharum spp.), a critical crop for sugar and bioenergy production, faces challenges in genetic improvement due to limited genetic diversity from selective breeding. Expanding genetic resources through intergeneric hybridization, particularly with Narenga porphyrocoma, offers a promising avenue to introduce traits like stress resistance and high biomass productivity. However, verifying true hybrids remains challenging with traditional morphological methods. This study employed tetra-primer ARMS-PCR and genomic in situ hybridization (GISH) to accurately identify intergeneric hybrids between S. officinarum and N. porphyrocoma. Species-specific primers were designed based on SNPs in the nrDNA-ITS region for ARMS-PCR, enabling effective differentiation of parental and hybrid genotypes, while GISH confirmed the chromosomal composition of hybrids, revealing an n + n inheritance pattern. The results demonstrated the potential of N. porphyrocoma to improve sugarcane’s tillering and leaf length, although sucrose content was lower in hybrids, suggesting the need for further breeding efforts. This study uniquely contributes to sugarcane breeding by providing an effective method for hybrid verification and laying a foundation for incorporating beneficial N. porphyrocoma genes into sugarcane cultivars.

Background Chromosomal inversion polymorphisms have been associated with adaptive behavioral, physiological, morphological and life history traits in the two main Afrotropical malaria vectors, Anopheles coluzzii and Anopheles gambiae . The understanding of the adaptive value of chromosomal inversion systems is constrained by the feasibility of cytological karyotyping. In recent years in silico and molecular approaches have been developed for the genotyping of most widespread inversions (2La, 2Rb and 2Rc). The 2Ru inversion, spanning roughly 8% of chromosome 2R, is commonly polymorphic in West African populations of An. coluzzii and An. gambiae and shows clear increases in frequency with increasing rainfall seasonally and geographically. The aim of this work was to overcome the constraints of currently available cytological and high-throughput molecular assays by developing a simple PCR assay for genotyping the 2Ru inversion in individual specimens of both mosquito species. Methods We designed tetra-primer amplification refractory mutation system (ARMS)-PCR assays based on five tag single-nucleotide polymorphisms (SNPs) previously shown to be strongly correlated with 2Ru inversion orientation. The most promising assay was validated against laboratory and field samples of An. coluzzii and An. gambiae karyotyped either cytogenetically or molecularly using a genotyping-in-thousands by sequencing (GT-seq) high-throughput approach that employs targeted sequencing of multiplexed PCR amplicons. Results A successful assay was designed based on the tag SNP at position 2R, 31710303, which is highly predictive of the 2Ru genotype. The assay, which requires only one PCR, and no additional post-PCR processing other than electrophoresis, produced a clear banding pattern for 98.5% of the 454 specimens tested, which is a 96.7% agreement with established karyotyping methods. Sequences were obtained for nine of the An. coluzzii specimens manifesting 2Ru genotype discrepancies with GT-seq. Possible sources of these discordances are discussed. Conclusions The tetra-primer ARMS-PCR assay represents an accurate, streamlined and cost-effective method for the molecular karyotyping of the 2Ru inversion in An. coluzzii and An. gambiae. Together with approaches already available for the other common polymorphic inversions, 2La, 2Rb and 2Rc, this assay will allow investigations of the adaptive value of the complex set of inversion systems observed in the two major malaria vectors in the Afrotropical region. Graphical Abstract

Leptosphaeria maculans is the causal agent of blackleg disease in canola (Brassica napus), resulting in significant yield loss in canola fields worldwide. AvrLm4-7 is an avirulence effector gene in L. maculans, and a single nucleotide mutation at codon 358 is responsible for the absence of the AvrLm4 allele. A tetra-primer amplification refractory mutation system-PCR assay (ARMS-PCR) was developed to rapidly differentiate the AvrLm4AvrLm7 and avrLm4AvrLm7 genes of L. maculans isolates, which differ by a single point mutation. By this approach, we were able to amplify distinct PCR products to infer the gene of the tested isolates. These results were also confirmed through phenotyping, using the cotyledon inoculation test and two canola genotypes with the corresponding resistance genes. The tetra-primer ARMS-PCR assay developed in this study is a simple, rapid, and useful protocol to identify the AvrLm4-7 alleles in L. maculans isolates. This assay has potential applications in the selection of resistant canola cultivars as part of broader antifungal strategies.

Background The present study aimed to determine the underlying genetic factors causing the possible Warburg micro syndrome (WARBM) phenotype in two Iranian patients. Case presentation A 5-year-old female and a 4.5-year-old male were referred due to microcephaly, global developmental delay, and dysmorphic features. After doing neuroimaging and clinical examinations, due to the heterogeneity of neurodevelopmental disorders, we subjected 7 family members to whole-exome sequencing. Three candidate variants were confirmed by Sanger sequencing and allele frequency of each variant was also determined in 300 healthy ethnically matched people using the tetra-primer amplification refractory mutation system-PCR and PCR-restriction fragment length polymorphism. To show the splicing effects, reverse transcription-PCR (RT-PCR) and RT-qPCR were performed, followed by Sanger sequencing. A novel homozygous variant—NM_012233.2: c.151-5 T > G; p.(Gly51IlefsTer15)—in the RAB3GAP1 gene was identified as the most likely disease-causing variant. RT-PCR/RT-qPCR showed that this variant can activate a cryptic site of splicing in intron 3, changing the splicing and gene expression processes. We also identified some novel manifestations in association with WARBM type 1 to touch upon abnormal philtrum, prominent antitragus, downturned corners of the mouth, malaligned teeth, scrotal hypoplasia, low anterior hairline, hypertrichosis of upper back, spastic diplegia to quadriplegia, and cerebral white matter signal changes. Conclusions Due to the common phenotypes between WARBMs and Martsolf syndrome (MIM: 212720), we suggest using the “RABopathies” term that can in turn cover a broad range of manifestations. This study can per se increase the genotype-phenotype spectrum of WARBM type 1.

Sugarcane is an important sugar and energy crop in the world. Germplasm innovation is a significant way to breed breakthrough sugarcane varieties. Modern sugarcane varieties all contain the blood relationship of Saccharum officinarum and Saccharum spontaneum. High sugar results from S. officinarum and the resistance genes from S. spontaneum. In order to improve the sugarcane quality, breeders use S. officinarum and S. spontaneum to cross and obtain hybrid offspring with high sugar and high resistance. Therefore, the authenticity of S. officinarum and S. spontaneum progeny materials directly affects the efficiency of sugarcane breeding. In this study, the tetra-primer amplification hindered mutation system (ARMS PCR) was used to identify ten suspected S. officinarum and eleven suspected S. spontaneum germplasm materials, then further validated by chromosome counting and genome in situ hybridization (GISH). Among the ten suspected S. officinarum materials to be identified, three were real S. officinarum materials, they were 14NG124, 51NG103, and Guan A. Nine of the eleven suspected S. spontaneum to be identified were fake S. spontaneum materials, these were Yunge 2007-12-165, Guangxi 87-20, Yunnan 82-16, Yunge 2007-11, YNLC 16, Laos No. 2, Yunnan 82-29, 2015-83, and 2013-20. The ARMS PCR results were the same as the GISH results. The three real S. officinarum materials had 80 chromosomes. Using ARMS PCR and GISH, three S. officinarum and nine S. spontaneum materials were proven to be authentic. Through chromosome number statistics, it was found that the three real S. officinarum had 80 chromosomes. Authentic materials were identified and selected to enrich the genetic background of sugarcane through hybridization and reduce the influence on the breeding process of the misuse of fake S. officinarum and S. spontaneum.

Background Prostatic adenocarcinoma is the most frequent malignancy among elderly men after lung cancer, which has the second incidence and the fourth mortality rate in the Iranian population. The primary objective of this study was to investigate how single-nucleotide polymorphisms of the CDH1 gene (rs16260) and DAB2IP (rs1571801) are associated with the risk of prostate cancer through a multi-stage approach. Results In the first stage of the study (58 men), we compared the genotype frequency of polymorphisms rs16260 and rs1571801 in the case group to the control group to determine significant polymorphisms (P value < 0.4). No statistically significant difference was shown between the genotype frequency of rs1571801 in the case and control groups. Thus, rs1571801 polymorphism was eliminated at this stage, and only rs16260 polymorphism evaluated in the next stage. In the second stage, statistical analysis showed a significant difference between genotype frequency of rs16260 (P value = 0.037) in all participants. The effect of rs16260 on prostate cancer was not modified by age or PSA levels. Only the Gleason Score = 7 reveals a significant difference between the risk allele (A) and the allele (C) (rs16260). Conclusions According to the results of this study, rs16260 is associated with prostate cancer predisposition and might be used as a potential biomarker in prostate cancer. It should be noted that these results need to be confirmed in a larger population.

Soybean (Glycine max L.) is gaining in importance due to its many uses, including as a food crop and a source of industrial products, among others. Increasing efforts are made to accelerate soybean research and develop new soybean varieties to meet global demands. Soybean research, breeding, identification, and variety protection all rely on precise genomic information. While DNA markers are invaluable tools for these purposes, the older generations, especially those developed before the advent of genome sequencing, lack precision and specificity. Thankfully, advancements in genome sequencing technologies have generated vast amounts of sequence data over the past decade, allowing precise and high-resolution analyses. However, making sense of the genomic information requires a certain level of professional training and computational power, which are not universally available to researchers. To address this, we generated a set of PCR-based DNA markers out of the existing genomic data from 228 popular soybean varieties that offer precise, unambiguous genomic information and can be easily adapted in various applications. A standard operating procedure (SOP) was also designed for these markers and validated on diverse soybean varieties to ensure their reproducibility. This user-friendly universal panel of DNA markers, along with the SOP, will facilitate soybean research and breeding programs through simple applications.

Introduction: End-stage renal disease (ESRD) is associated with critical kidney illness that seriously affects the lifespan. Genetic factors and oxidative stress could play critical role in the development of ESRD. We assessed the association between chemerin rs17173608 T/G and vaspin rs2236242 T/A genes variants with the risk of ESRD and their correlation with plasma malondialdehyde (MDA) level. Materials and methods: In a case-control study, 131 gender and age-matched unrelated healthy controls and 110 ESRD patients were enrolled. The chemerin rs17173608 T/G and vaspin rs2236242 T/A were detected by Tetra primer-amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR). The MDA concentration was determined by HPLC. Results: Our findings for the first time revealed that in codominant genetic model (T/G vs. T/T genotype), the T/G genotype of chemerin gene significantly had a protective role against ESRD susceptibility. Also, in the presence of chemerin G allele, the risk of ESRD decreased by 0.79-fold (p = .048) in Kurdish population of Iran. The MDA serum levels in ESRD patients carrying the chemerin T/G + G/G genotype of rs17173608 T/G and also in carriers of A/A + T/A genotype of vaspin rs2236242 T/A were significantly higher compared to those in control subjects. The overall distribution of vaspin rs2236242 T/A genotypes and alleles comparing ESRD patients and healthy subjects were not statistically significant. Conclusion: We found that the G allele of chemerin rs17173608 compared to T allele decreased the risk of ESRD, and there was a significant association between chemerin and vaspin variants with plasma MDA level in a sample of the Iranian population.