Allele-specific PCR Research Articles

The role of Toll-like receptor 4 Asp299Gly genetic polymorphism in rehabilitation of patients with post-COVID syndrome

Introduction. The multivariability of the clinical course of post-covid syndrome (PCS) is associated with genetic heterogeneity of the population, which leads to a high interest in the study of genotypes. Aim. To evaluate the role of Toll-like receptor 4 (TLR4 (Asp229Gly) polymorphism in the formation of PCS and rehabilitation potential. Materials and methods. The study included 92 patients diagnosed with PCS between the ages of 21 and 75. All patients received a comprehensive clinical, laboratory, functional examination and quality of life assessment at admission and discharge. Deoxyribonucleic acid isolation and allele-specific polymerase chain reaction were performed from whole blood using «Lytech» kits (Russia) according to the manufacturer's instructions. Formation of groups for statistical processing was carried out in accordance with the results of genetic analysis. Patients received comprehensive rehabilitation, including: climatotherapy, exercise therapy, massage, aromatherapy, respiratory therapy. Performance was assessed based on the dynamics of the studied indicators. Results and discussion. Of the 92 patients included in the study, 22 (23.9 %) were AA homozygotes, 33 (35.8 %) were GG, and 37 (40.3 %) were AG heterozygotes. By age, the groups were comparable. The gender ratio was predominantly female in all groups. According to the initial characteristics, the condition of patients with more pronounced clinical symptoms was in the group of patients with the AG genotype. All groups showed varying degrees of positive dynamics. The dependence of the severity of PCS and the effectiveness of rehabilitation on genetic predisposition was established. Different severity of initial clinical symptoms and deviations of functional parameters were revealed. Significant differences were found when assessing the effectiveness of rehabilitation. Positive dynamics was observed in all groups, but most successfully in the AG group. This confirms the role of TLR4 in the immunological response to SARS-CoV-2 and its genetic polymorphism in a wide range of manifestations of both acute COVID-19 and PCS. Conclusions. The effectiveness of rehabilitation in patients with PCS is interrelated with the genetic heterogeneity of TLR4. The heterozygous variant of the AG genotype forms more pronounced clinical manifestations of PCS, but also a higher rehabilitation potential, which contributes to the successful completion of rehabilitation. The minor allele TLR4 (Asp299Gly) G is associated with a worsening of the course of PCS. Patients with the homozygous GG genotype need a longer course of rehabilitation.

Variations in HBA gene contribute to high-altitude hypoxia adaptation via affected O2 transfer in Tibetan sheep

Tibetan sheep are indigenous to the Qinghai-Xizang Plateau. Owing to the harsh hypoxic environment in this plateau, the hemoglobin (Hb) protein in Tibetan sheep has undergone adaptive changes over time. Hb is primarily responsible for transporting O2 and CO2 between the lungs and other tissues of the body. The α subunit of Hb, encoded by the HBA gene, is a crucial component of the protein. However, whether variations in the HBA gene sequence affect the adaptation of Tibetan sheep to high-altitude hypoxia remains unclear. In this study, we sequenced the HBA gene and identified three single nucleotide polymorphisms (SNPs). These SNPs were genotyped in Tibetan and Hu sheep using Kompetitive Allele-Specific PCR (KASP). The results showed that the frequencies of the AT genotype and H1H2 haplotype were higher in Tibetan sheep than in Hu sheep. Individuals with the AT genotype exhibited higher P50 levels, whereas those with the H1H2 haplotype exhibited lower PO2 and SaO2 levels. The higher P50 levels indicated that O2 was more readily released from oxygenated Hb into the tissues, with the lower PO2 and SaO2 levels facilitating this process. These findings indicate that variations in the HBA gene sequence contribute to enhancing O2 transfer efficiency in Tibetan sheep.

Allelic variation at the PPD-A1 locus and its associations with heading time and winter wheat (Triticum aestivum L.) agronomic traits in the northern Black Sea region

Aim. The identification of Ppd-1-alleles in winter bread wheat varieties of various origin, including the ones of Ukrainian plant breeding and recombinant-inbred lines Orenburgskaya 48//Cappelle Desprez/2В Chinese Spring, and the evaluation of the effects of allele Ppd-А1_del303, including the interaction with different alleles of gene Ppd-B1, by the duration of the period before heading and the related agronomically valuable traits. Methods. DNA extraction, allele-specific PCR, agarose and polyacrylamide gel electrophoresis, phenological observations, evaluation of frost resistance in seedlings, and analysis of morphobiological traits and elements of yield structure. Statistical analysis of the obtained data was carried out in Microsoft Excel. The significance of the difference between samples was assessed by Fisher's F test. A difference of p < 0.05 was considered statistically significant for all indicators. Results. The marking of 30 varieties of different origin and 64 recombinant-inbred lines of Orenburgskaya 48//Cappelle Desprez/2B Chinese Spring winter bread wheat was carried out to identify the alleles of the Ppd-A1 gene. The polymorphism of varieties and populations of recombinant-inbred lines in the northern Black Sea region (Odesa) was evaluated for ten traits: frost resistance of plants in the seedling phase, winter hardiness, duration of the period before heading, plant height, grain number per spike, grain weight per spike, thousand grain weight, number of productive tillers per unit area, harvest index and grain yield. The comparison of the lines evaluation data in terms of agronomic traits and the results of the genotypes identification allowed us to identify the influence of Ppd-A1 gene alleles and various combinations of the alleles of Ppd-A1 and Ppd-B1 genes on these traits. Conclusions. A higher prevalence of the Ppd-A1_del303 allele was found both among varieties and recombinant-inbred lines. The genetic differences by the Ppd-A1 gene (Ppd-A1_del303 or Ppd-A1b) are significantly related only to frost resistance of seedlings in the absence of significant differences in other traits. The interaction between Ppd-A1b and Ppd-B1c alleles contributed to the acceleration of early maturity and the formation of the highest indicators of grain weight per spike, thousand grain weight, harvest index, and grain yield. The replacement of the Ppd-A1b allele with Ppd-A1_del303 led to a decrease in the effect of the dominant Ppd-B1c allele on accelerating heading and negatively affected the grain weight per spike, thousand grain weight, harvest index and grain yield compared to the Ppd-A1b Ppd-B1c genotype.

Identification and validation of two quantitative trait loci showing pleiotropic effect on multiple grain-related traits in bread wheat (Triticum aestivum L.).

QKl/Tgw/Gns.yaas-2D associates with KL, TGW, and GNS, and QKl/Tgw.yaas-5A associates with KL and TGW. Significantly pleiotropic and additive effects of these two QTL were validated. The YM5 allele both at QKl/Tgw/Gns.yaas-2D and QKl/Tgw.yaas-5A was proved to be the best allelic combination for improving yield potential. Kernel length (KL), kernel width (KW), thousand grain weight (TGW), and grain number per spike (GNS) play important roles in the yield improvement of wheat. In this study, one recombinant inbred line (RIL) derived from a cross between Yangmai 5 (YM5) and Yanzhan 1 (YZ1) was used to identify quantitative trait loci (QTL) associated with KL, KW, TGW, and GNS across three years. Two pleiotropic QTL namely QKl/Tgw/Gns.yaas-2D and QKl/Tgw.yaas-5A were located in two genomic regions on chromosomes 2D and 5A, respectively. Breeder-friendly Kompetitive Allele-Specific PCR (KASP) markers for QKl/Tgw/Gns.yaas-2D and QKl/Tgw.yaas-5A were developed and validated in a set of 246 wheat cultivars/lines. Analysis of allelic combinations indicated that the YM5 allele both at QKl/Tgw/Gns.yaas-2D and QKl/Tgw.yaas-5A is probably the best one to promote TGW, GNS, and grain weight per spike. Based on the analysis of gene annotation, sequence variations, expression patterns, and GO enrichment, twenty-five and twenty-four candidate genes of QKl/Tgw/Gns.yaas-2D and QKl/Tgw.yaas-5A, respectively, were identified. These results provide the basis of fine-mapping the target QTL and marker-assisted selection in wheat yield-breeding programs.

Detection of Candidate Genes and Development of KASP Markers for Pod Length and Pod Width by Combining Genome-Wide Association and Transcriptome Sequencing in Vegetable Soybean

Vegetable soybeans are one of the most important vegetable types in East Asia. The yield of vegetable soybeans is considerably influenced by the size of their pods. To facilitate the understanding of the genetic basis of the pod length and width in vegetable soybeans, we conducted a genome-wide association study (GWAS) and transcriptome sequencing. Four quantitative trait loci, namely, qGPoL1, qGPoL2, qGPoW1, and qGPoW2, were mapped via GWAS analysis. Through the integration of gene function annotation, transcriptome sequencing, and expression pattern analysis, we identified Glyma.06G255000 and Glyma.13G007000 as the key determinants of the pod length and width in vegetable soybeans, respectively. Furthermore, two kompetitive allele-specific polymerase chain reaction (KASP) markers, namely, S06-42138365 (A/T) and S13_628331 (A/T), were developed and effectively validated in 27 vegetable soybean accessions. Overall, our research identified genes that regulate the pod length and width and determined KASP markers for molecular marker-assisted selection breeding. These findings have crucial implications for the improvement of soybean crops and can contribute to the development of efficient breeding strategies.

Characterization of a new barley greenbug resistance gene Rsg4 in the Chinese landrace CI 2458.

Barley (Hordeum vulgare) is a climate-resilient crop widely cultivated in both highly productive and suboptimal agricultural systems, and its ability to adapt to multiple biotic and abiotic stresses has contributed significantly to food security. Greenbug is a destructive insect pest for global barley production, and new greenbug resistance genes are needed to overcome the challenges posed by diverse greenbug biotypes in fields. CI 2458 is a Chinese landrace exhibiting a unique resistance profile to a set of 14 greenbug biotypes, which suggests the presence of a new greenbug resistance gene in CI 2458. A recombinant inbred line population from the cross Weskan × CI 2458 was developed, evaluated for responses to greenbug biotype F, and genotyped using single nucleotide polymorphism (SNP) markers generated by genotyping-by-sequencing. Linkage analysis revealed a single gene, designated Rsg4, conditioning greenbug resistance in CI 2458. Rsg4 was delimited to a 1.14 Mb interval between SNP markers S3H_666512114 and S3H_667651446 in the terminal region of chromosome arm 3HL, with genetic distances of 1.2 cM proximal to S3H_667651446 and 1.1 cM distal to S3H_666512114. Allelism tests confirmed that Rsg4 is a new greenbug resistance gene independent of Rsg1 and Rsg3, which reside in the same chromosome arm. Rsg4 differs from Rsg1 alleles and Rsg3 in its resistance to greenbug biotype TX1, one of the most widely virulent biotypes. The introgression of Rsg4 into locally adapted barley cultivars is of agronomic importance, and kompetitive allele-specific polymerase chain reaction (KASP) markers flanking Rsg4, KASP-Rsg336-1 and KASP-Rsg336-2, enable rapid pyramiding of Rsg4 with other resistance genes to develop durable greenbug-resistant cultivars.

Fine mapping of QGPC.caas-7AL for grain protein content in bread wheat.

A major stable QTL, QGPC.caas-7AL, for grain protein content of wheat, was narrowed down to a 1.82-Mb inter on chromosome 7AL, and four candidate genes were predicated. Wheat grain protein content (GPC) is important for end-use quality. Identification of genetic loci for GPC is helpful to create new varieties with good processing quality and nutrients. Zhongmai 578 (ZM578) and Jimai 22 (JM22) are two elite wheat varieties with different contents of GPC. In the present study, 262 recombinant inbred lines (RILs) derived from a cross between ZM578 and JM22 were used to map the GPC with high-density wheat Illumina iSelect 50K single-nucleotide polymorphism (SNP) array. Seven quantitative trait loci (QTLs) were identified for GPC on chromosomes 3AS, 3AL, 3BS, 4AL, 5BS, 5DL and 7AL by inclusive composite interval mapping, designated as QGPC.caas-3AS, QGPC.caas-3AL, QGPC.caas-3BS, QGPC.caas-4AL, QGPC.caas-5BS, QGPC.caas-5DL and QGPC.caas-7AL, respectively. Among these, alleles for increasing GPC at QGPC.caas-3AS, QGPC.caas-3BS, QGPC.caas-4AL and QGPC.caas-7AL loci were contributed by ZM578, whereas those at the other three loci were from JM22. The stable QTL QGPC.caas-7AL was fine mapped to a 1.82-Mb physical interval using secondary populations from six heterozygous recombinant plants obtained by selfing a residual RIL. Four genes were predicted as candidates of QGPC.caas-7AL based on sequence polymorphism and expression patterns. The near-isogenic lines (NILs) with the favorable allele at the QGPC.caas-7AL locus increased Farinograph stability time, Extensograph extension area, extensibility and maximum resistance by 19.6%, 6.3%, 6.0% and 20.3%, respectively. Kompetitive allele-specific PCR (KASP) marker for QGPC.caas-7AL was developed and validated in a diverse panel of 166 Chinese wheat cultivars. These results provide further insight into the genetic basis of GPC, and the fine-mapped QGPC.caas-7AL will be an attractive target for map-based cloning and marker-assisted selection in wheat breeding programs.

Target-site mediated insecticide resistance in major mosquito (Diptera: Culicidae) vectors: A systematic review

The use of insecticides remain the most effective vector control approach for mosquito borne diseases like malaria, dengue fever, chikungunya, and Zika virus disease; however, their increasing resistance has complicated the management. Mutations in kdr, ace-1/ace-2, rdl, and nAChR, are involved in target-site mediated resistance and prevent the binding of pyrethroids and dichlorodiph enyltrichloroethane, organophosphorus pesticide, carbamates and cyclodienes, respectively. Here, we review the current knowledge on target-site mediated insecticidal resistance mechanisms in major mosquito vectors and the techniques used to identify these mutations. The results showed that kdr mutations are frequently reported in mosquito species, indicating the overuse of pyrethroid in mosquito control. Report on ace mutations is very limited, primarily detected in Anopheles and Culex, with extremely low detection rate in Aedes, despite the wide application of organophosphates in their control. Notably, reports of rdl mutations and changes in nAChR are rare, which provides an opportunity to exploit these target sites, particularly in the event of disease outbreaks. Among various detection methods such as RFLP, TaqMan, Multiplex PCR and FRET, allele-specific PCR is mostly employed, especially in detecting kdr mutation. However, allele-specific PCR cannot detect novel mutation. As a result, there is an urgent need to develop a scalable, cost-effective tool that can be widely applied especially as these mutations are genetic markers for early detection of insecticide resistance. Robust monitoring methods remain critical to manage insecticide resistance and effective control of mosquito-borne diseases.

Identification and molecular marker development for peel color gene in melon (Cucumis melo L.)

Peel color is an important appearance quality of melons that greatly affects consumer preferences. In this study, a near-isogenic line NIL-G (dark green peel) was generated from B8 (grey-green peel) and B15 (white peel). The F2 population constructed by crossing NIL-G and B15 was used to study the inheritance pattern of peel color, and bulked-segregant analysis sequencing (BSA-seq) was employed to identify the interval in which the target gene was located. Genetic analysis results showed that the dark green peel trait at maturity is controlled by a dominant gene. BSA-seq and molecular markers were used to localize the candidate gene in a 263.7 kb interval of chromosome 4, which contained the CmAPRR2 gene with known functions. Moreover, allelic sequence analysis revealed four SNP variations of the CmAPRR2 gene in B15, of which SNP.G614331A was located at the junction of the 6th exon and 6th intron. The G-to-A mutation caused alternative splicing of the transcript of CmAPRR2 in B15, generating two transcripts (CmAPRR2-A and CmAPRR2-B) with premature termination codons. Furthermore, the Kompetitive Allele Specific PCR (KASP) marker, APRR2-G/A, was developed based on this SNP and shown to co-segregate with the peel color phenotype in the F2 population. Compared to white-peel B15, the expression level of CmAPRR2 in dark green peel NIL-G was higher at each growth stage. Therefore, CmAPRR2 may be the key gene controlling the fruit color of melons. Overall, this study identified a novel allelic variant of CmAPRR2 that leads to white peel formation in mature melons. The study also provides a theoretical basis for further research on the gene regulatory mechanism of melon peel colors and may promote the future use of molecular marker-assisted selection to modify melon peel colors.

Combined bulked segregant analysis and Kompetitive Allele-Specific PCR genotyping identifies candidate genes related to the node of the first fruiting branch in upland cotton (Gossypium hirsutum L.)

Combined bulked segregant analysis and Kompetitive Allele-Specific PCR genotyping identifies candidate genes related to the node of the first fruiting branch in upland cotton (Gossypium hirsutum L.)

Rapid detection of mutations in the suspected piperaquine resistance gene E415G-exo in Plasmodium falciparum exonuclease via AS‒PCR and RAA with CRISPR/Cas12a

Malaria remains a major public health concern. The rapid spread of resistance to antimalarial drugs is a major challenge for malaria eradication. Timely and accurate molecular monitoring based on practical detection methods is a critical step toward malaria control and elimination. In this study, two rapid detection techniques, allele-specific PCR (AS‒PCR) and recombinase-aided amplification (RAA) combined with CRISPR/Cas12a, were established, optimized and assessed to detect single nucleotide polymorphisms in the Plasmodium falciparum exonuclease (Pfexo) gene related to suspected piperaquine resistance. Moreover, phosphorothioate and artificial mismatches were introduced into the allele-specific primers for AS‒PCR, and crRNA-mismatched bases were introduced into the RAA‒CRISPR/Cas12a assay because crRNAs designed according to conventional rules fail to discriminate genotypes. As a result, the detection limits of the AS‒PCR and RAA‒CRISPR/Cas12a assays were 104 copies/μL and 103 copies/μL, respectively. The detection threshold for dried blood spots was 100‒150 parasites/μL, with no cross-reactivity against other genotypes. The average cost of AS‒PCR is approximately $1 per test and takes 2–3 h, whereas that of the RAA‒CRISPR/Cas12a system is approximately $7 per test and takes 1 h or less. Therefore, we provide more options for testing single nucleotide polymorphisms in the Pfexo gene, considering economic conditions and the availability of instruments, equipment, and reagents, which can contribute to the molecular monitoring of antimalarial resistance.

QTaHa-5DL: another major QTL regulating wheat grain hardness

Grain hardness has important effects on grain quality and the end-use of wheat. In this study, a collection of 103 common wheat germplasms and a DH population of 194 lines were used to identify new quantitative trait loci (QTL) for grain hardness. Two stable genetic loci on chromosome 5D were detected under different locations and years with one of them being the Ha locus on 5DS where the major gene Puroindolines for wheat grain hardness is located. Another locus of qTaHa-5DL also showed a significant impact on grain hardness index (HI) with HI increasing from ~ 20 to ~ 45 and hardness type changing from soft to mixed when Puroindolines are wild type. A kompetitive allele-specific PCR (KASP) marker for qTaHa-5DL was developed and the effectiveness of the QTL was confirmed in 184 breeding lines with the marker K-Ha5DL dividing wheat into two distinct categories. This new QTL can be effectively used to select soft or medium hard wheat.

Identification of diagnostic challenges in RP1 Alu insertion and strategies for overcoming them

Recently, a founder Alu insertion in exon 4 of RP1 was detected in Japanese and Korean patients with inherited retinal diseases (IRDs). However, carrier frequency and diagnostic challenges for detecting AluY insertion are not established. We aim to investigate the frequency of AluY in individuals with or without IRDs and to overcome common diagnostic pitfalls associated with AluY insertion. A total of 1,072 subjects comprising 411 patients with IRD (IRD group) and 661 patients with other suspected Mendelian genetic disease (non-IRD group) was screened for AluY insertion. Targeted panel sequencing and whole-genome sequencing were used for detection of AluY insertion, and an optimized allele-specific PCR (AS-PCR) was used for validation. The AluY insertion was detected in 1.5% in IRD group (6/411). The AluY insertion was not observed in non-IRD group (0/661). All patients with AluY were confirmed to have RP1 pathogenic variants on the paired allele. We identified AluY allele dropout leading to false homozygosity for c.4196del pathogenic variant in Sanger sequencing. The allelic relationship between variants of RP1 was accurately determined by AluY AS-PCR. Delineating diagnostic challenges of AluY insertion and strategies to avoid potential pitfalls could aid clinicians in an accurate molecular diagnosis for patients with IRD.

Genome-wide mapping of quantitative trait loci conferring resistance to stripe rust in spring wheat line PI 660072.

Two major QTL for resistance to stripe rust were mapped on chromosome 2BL and 4BL in spring wheat PI 660072, and their KASP markers were developed. Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most devastating diseases of wheat worldwide. Identifying resistance genes is crucial for developing resistant cultivars to control the disease. Spring wheat PI 660072 (Triticum aestivum) has been identified to possess both adult-plant resistance (APR) and all-stage resistance (ASR) to stripe rust. To elucidate the genetic basis of the resistance in PI 660072, a mapping population consisting of 211 F5-F7 recombinant-inbred lines (RILs) was developed from a cross of PI 660072 with susceptible spring wheat Avocet S. The mapping population was phenotyped for stripe rust responses across five field environments from 2020 to 2022 and genotyped using the 15K SNP (single nucleotide polymorphism) array to map stripe rust resistance loci. The mapping population was also tested at the seedling stage with predominant Chinese Pst races CYR31, CYR32, CYR34 and PST-YX1-3-1 in the greenhouse. Stripe rust resistance genes were identified using the quantitative trait locus (QTL) mapping approach. Two QTL were identified with QYrPI660072.swust-2BL mapped on the long arm of chromosome 2B for ASR and QYrPI660072.swust-4BL on the long arm of chromosome 4B for APR. To facilitate marker-assisted selection breeding, Kompetitive allele specific PCR (KASP) markers, KASP-1269 for QYrPI660072.swust-2BL and KASP-3209 for QYrPI660072.swust-4BL, were developed. These markers could be used to introgress the effective resistance QTL into new wheat cultivars.

Genome-wide association study (GWAS) analyses of early anatomical changes in rose adventitious root formation

Adventitious root (AR) formation is a genetically complex trait with high genotypic variability. Therefore, only a limited range of cultivars are currently propagated by cuttings in rose. In this study, we analysed the anatomy of in vitro shoots, the early formation of root primordia (RP) and the formation of ARs in a diverse set of 106 rose genotypes. Correlation analysis indicated that the growth in shoot diameter and the vasculature dimensions after 1 week of rooting contributed to successful AR formation. Using phenotypic data for genome-wide association studies (GWAS) analyses, nine significantly associated single nucleotide polymorphisms (SNPs) and genomic regions contributing to various RP and AR formation traits were identified. The contribution of genomic regions to trait variation was notably greater for traits associated with earlier processes than for traits associated with later developmental stages. The combination of RP and AR data allowed the detection of regions by GWAS that contain factors that potentially limit RP emergence. Homologues of 47 genes known to be involved in AR formation from the literature could be assigned to the identified peaks. Further studies are needed to investigate the suitability of SNPs exhibiting strong effects as allele-specific PCR markers for use in breeding.

Genome-wide association studies in a diverse strawberry collection unveil loci controlling agronomic and fruit quality traits.

Strawberries (Fragaria sp.) are cherished for their organoleptic properties and nutritional value. However, breeding new cultivars involves the simultaneous selection of many agronomic and fruit quality traits, including fruit firmness and extended postharvest life. The strawberry germplasm collection here studied exhibited extensive phenotypic variation in 26 agronomic and fruit quality traits across three consecutive seasons. Phenotypic correlations and principal component analysis revealed relationships among traits and accessions, emphasizing the impact of plant breeding on fruit weight and firmness to the detriment of sugar or vitamin C content. Genetic diversity analysis on 124 accessions using 44,408 markers denoted a population structure divided into six subpopulations still retaining considerable diversity. Genome-wide association studies for the 26 traits unveiled 121 significant marker-trait associations distributed across 95 quantitative trait loci (QTLs). Multiple associations were detected for fruit firmness, a key breeding target, including a prominent locus on chromosome 6A. The candidate gene FaPG1, controlling fruit softening and postharvest shelf life, was identified within this QTL region. Differential expression of FaPG1 confirmed its role as the primary contributor to natural variation in fruit firmness. A kompetitive allele-specific PCR assay based on the single nucleotide polymorphism (SNP) AX-184242253, associated with the 6A QTL, predicts a substantial increase in fruit firmness, validating its utility for marker-assisted selection. In essence, this comprehensive study provides insights into the phenotypic and genetic landscape of the strawberry collection and lays a robust foundation for propelling the development of superior strawberry cultivars through precision breeding.

Report of 3 Cases Diagnosed with Basal Cell Carcinoma and Systemic Mastocytosis with Favorable Prognosis

Abstract Introduction/Objective The co-existence of basal cell carcinoma (BCC) and systemic mastocytosis is an exceptionally rare clinical occurrence, and the prognosis is not well-documented. Understanding the clinical and histopathological features of this uncommon association is relevant for accurate diagnosis, management, and prognostication. Methods/Case Report We conducted a retrospective analysis of three cases of BCC with concurrent systemic mastocytosis that were identified in the pathology database of the University of Pennsylvania from 2010 to 2023. Patient demographics, clinical and histopathologic characteristics, and treatment outcomes were reviewed. Histopathologic assessment, immunohistochemistry, and C-KIT D816V mutation analysis (with an allele-specific PCR assay) were performed on biopsy material. Next-Generation Sequencing (NGS) was used to identify other hematologic neoplasm-associated mutations. Results (if a Case Study enter NA) All three cases were female with a median age of 55.3 years (50, 69, 47). In all three cases, atypical CD117+ tryptase+ CD25+ mast cells with mild to moderate pleomorphism were found in the dermis adjacent to the BCC cells. All three cases had bone marrow involvement. Serum tryptase levels were mildly elevated in all 3 patients (maximum 65.5 μg/L). All three cases harbor a C-KIT D816V mutation and a history of systemic glucocorticosteroids before the diagnosis of BCC. One of the cases was treated with phototherapy. NGS detected the following somatic mutations: Case #2 DNMT3A p.R882H c.2645G>A; Case #3 JAK2 V617F. Case #1 had multiple relapses of BCC, but a subsequent marrow biopsy did not identify any involvement by mastocytosis. Case #2 had mast cell leukemia, which typically has a poor prognosis; however, this patient has survived for several years. Case #3 had essential thrombocythemia and systemic mastocytosis with an associated hematological neoplasm, and her symptoms were stable. Treatment modalities for the three patients ranged from surgical excision to anti-mastocytosis therapy, with favorable outcomes (Table 1). Conclusion The coexistence of BCC with mastocytosis poses diagnostic challenges due to its infrequency and varied clinical presentations. Previous literature reports on treatments of mastocytosis such as phototherapy and steroids, which have been identified as risk factors for the development of basal cell carcinoma. However, recognizing this unique combination in clinical practice may aid in providing appropriate treatment strategies, ultimately leading to favorable patient outcomes.

Development of a simple allele-specific PCR for the detection of pathogenic Vibrio cholerae O1 and O139 in seafood

This study developed a simple allele specific (AS) PCR assay for the detection of pathogenic Vibrio cholerae strains in seafood. A pair of primers, (1) an AS primer targeting the mutation at site G450 in the hapR2 allele and a (2) common forward primer, were designed to detect the pathogenic O1 and O139 strains. An artificial mismatch was also added to the AS primer to increase its specificity to the target allele. The PCR assays using different DNA polymerases of varying efficiencies were then optimized. The application of the AS primers in both conventional and real-time PCR assays was facilitated with enriched seafood samples spiked with known concentration of pathogenic V. cholerae. The designed AS primers showed high specificity and sensitivity to the hapR2 allele for both conventional and real-time PCR assays. Enrichment of seafood samples for 8 h is recommended to detect viable pathogenic V. cholerae presence in seafood. In this study, we developed an AS-PCR that is simple, rapid, and a low-cost alternative for routine detection of pathogenic V. cholerae in different seafood samples thus aiding in the surveillance and implementation of food safety measures at the seafood post-harvest level.

Identification of genetic loci for powdery mildew resistance in common wheat.

Powdery mildew (PM) poses an extreme threat to wheat yields and quality. In this study, 262 recombinant inbred lines (RILs) of Doumai and Shi 4185 cross were used to map PM resistance genes across four environments. High-density genetic linkage map of the Doumai/Shi 4185 RIL population was constructed using the wheat Illumina iSelect 90K single-nucleotide polymorphism (SNP) array. In total, four stable quantitative trait loci (QTLs) for PM resistance, QPm.caas-2AS, QPm.caas-4AS, QPm.caas-4BL, and QPm.caas-6BS, were detected and explained 5.6%-15.6% of the phenotypic variances. Doumai contributed all the resistance alleles of QPm.caas-2AS, QPm.caas-4AS, QPm.caas-4BL, and QPm.caas-6BS. Among these, QPm.caas-4AS and QPm.caas-6BS overlapped with the previously reported loci, whereas QPm.caas-2AS and QPm.caas-4BL are potentially novel. In addition, six high-confidence genes encoding the NBS-LRR-like resistance protein, disease resistance protein family, and calcium/calmodulin-dependent serine/threonine-kinase were selected as the candidate genes for PM resistance. Three kompetitive allele-specific PCR (KASP) markers, Kasp_PMR_2AS for QPm.caas-2AS, Kasp_PMR_4BL for QPm.caas-4BL, and Kasp_PMR_6BS for QPm.caas-6BS, were developed, and their genetic effects were validated in a natural population including 100 cultivars. These findings will offer valuable QTLs and available KASP markers to enhance wheat marker-assisted breeding for PM resistance.

Genome-wide identification of the endonuclease family genes implicates potential roles of TaENDO23 in drought-stressed response and grain development in wheat

BackgroundEndonucleases play a crucial role in plant growth and stress response by breaking down nuclear DNA. However, the specific members and biological functions of the endonuclease encoding genes in wheat remain to be determined.ResultsIn this study, we identified a total of 26 TaENDO family genes at the wheat genome-wide level. These genes were located on chromosomes 2 A, 2B, 2D, 3 A, 3B, and 3D and classified into four groups, each sharing similar gene structures and conserved motifs. Furthermore, we identified diverse stress-response and growth-related cis-elements in the promoter of TaENDO genes, which were broadly expressed in different organs, and several TaENDO genes were significantly induced under drought and salt stresses. We further examined the biological function of TaENDO23 gene since it was rapidly induced under drought stress and exhibited high expression in spikes and grains. Subcellular localization analysis revealed that TaENDO23 was localized in the cytoplasm of wheat protoplasts. qRT-PCR results indicated that the expression of TaENDO23 increased under PEG6000 and abscisic acid treatments, but decreased under NaCl treatment. TaENDO23 mainly expressed in leaves and spikes. A kompetitive allele-specific PCR (KASP) marker was developed to identify single nucleotide polymorphisms in TaENDO23 gene in 256 wheat accessions. The alleles with TaENDO23-HapI haplotypes had higher grain weight and size compared to TaENDO23-HapII. The geographical and annual frequency distributions of the two TaENDO23 haplotypes revealed that the elite haplotype TaENDO23-HapI was positively selected in the wheat breeding process.ConclusionWe systematically analyzed the evolutionary relationships, gene structure characteristics, and expression patterns of TaENDO genes in wheat. The expression of TaENDO23, in particular, was induced under drought stress, mainly expressed in the leaves and grains. The KASP marker of TaENDO23 gene successfully distinguished between the wheat accessions, revealing TaENDO23-HapI as the elite haplotype associated with improved grain weight and size. These findings provide insights into the evolution and characteristics of TaENDO genes at the genome-wide level in wheat, laying the foundation for further biological analysis of TaENDO23 gene, especially in response to drought stress and grain development.