Cross-Resistance of Succinate Dehydrogenase Inhibitors (SDHI) in Botrytis cinerea and Development of Molecular Diagnostic Tools for SDHI Resistance Detection.

Succinate dehydrogenase inhibitors (SDHIs) are keystone synthetic fungicides used to manage Botrytis cinerea in several hosts. In this study, we investigated the cross-resistance between five new SDHIs (pyraziflumid, isofetamid, benzovindiflupyr, fluxapyroxad, and pydiflumetofen) with commonly used SDHIs boscalid and fluopyram. Different mutations were detected in the sdhB gene in B. cinerea collected from Michigan grapes, and their frequency and EC50 value were determined. Among 216 B. cinerea boscalid-resistant isolates, five different mutations were detected, including H272R/Y, P225F/H, and N230I, at frequencies of 82.6, 4.3, 11.5, 0.4, and 5.3%, respectively. Five isolates of each genotype were used to screen the cross-resistance of the SDHIs. We classified the resistance profile of our mutants into five patterns. We report that all tested mutants were sensitive to benzovindiflupyr, indicating that it can be used as an effective fungicide against all B. cinerea mutants identified in this study. In addition, fluopyram, pydiflumetofen, and isofetamid can provide effective control according to which type of mutation is present in the field. We also developed and compared two molecular diagnostic tools, rhAMP and TaqMan assays, for rapid detection of SDHI resistance-associated mutants in B. cinerea. We report that the TaqMan assay was more successful than the rhAMP assay in detecting the B. cinerea mutant DNA at ≤10 pg and in a single assay was capable of monitoring two amino acid positions. Our results provide essential information about new SDHIs and provide molecular tools for monitoring SDHI resistance mutations, which will assist in gray mold disease control.

Genotype variation of ACE and ACE2 genes affects the severity of COVID-19 patients.

OBJECTIVE: Genetic polymorphisms in ACE and ACE2 genes are involved in the RAS regulation of blood pressure and their activity may confer susceptibility to hypertension. In addition, they may play a role in SARS-CoV-2 pathogenesis and the severity of COVID-19. This study aims to determine the effect of genetic variations in the ACE (rs4331) and ACE2 (rs2074192) genes with hypertension comorbidity on the severity of COVID-19 in the Indonesian population. RESULT: 186 patients were enrolled and assigned into the COVID-19 group (n = 95) and non-COVID-19 group (n = 91) in this cross-sectional study. GG genotype frequency was dominant in ACE gene, but there were no significant differences between the groups (p = 0.163). The two groups had a significant difference (p = 0.000) for the CC genotype frequency (0,37 vs. 0.01) in the ACE2 gene. The proportion of women with COVID-19 is higher (51%), but men with hypertension had more severe symptoms (44%). Men with hypertension comorbidity, GG (ACE), and TT (ACE2) genotypes tended to have moderate-to-severe symptoms (25%). Similarly, women with hypertension as well as GG and CT genotypes tended to have moderate-to-severe symptoms (21%). We conclude that hypertension and mutations in the ACE (rs4331) and ACE2 (rs2074192) genes affect the severity of COVID-19.

Genotyping tools and resources to assess peanut germplasm: smut-resistant landraces as a case study.

Peanut smut caused by Thecaphora frezii is a severe fungal disease currently endemic to Argentina and Brazil. The identification of smut resistant germplasm is crucial in view of the potential risk of a global spread. In a recent study, we reported new sources of smut resistance and demonstrated its introgression into elite peanut cultivars. Here, we revisited one of these sources (line I0322) to verify its presence in the U.S. peanut germplasm collection and to identify single nucleotide polymorphisms (SNPs) potentially associated with resistance. Five accessions of Arachis hypogaea subsp. fastigiata from the U.S. peanut collection, along with the resistant source and derived inbred lines were genotyped with a 48K SNP peanut array. A recently developed SNP genotyping platform called RNase H2 enzyme-based amplification (rhAmp) was further applied to validate selected SNPs in a larger number of individuals per accession. More than 14,000 SNPs and nine rhAmp assays confirmed the presence of a germplasm in the U.S. peanut collection that is 98.6% identical (P < 0.01, bootstrap t-test) to the resistant line I0322. We report this germplasm with accompanying genetic information, genotyping data, and diagnostic SNP markers.

New high-sensitive rhAmp method for A1 allele detection in A2 milk samples.

Cows' milk may contain two types of ß-casein: A1 and A2. A1 digestion is associated with the release of ß-casomorphine-7 peptide, which can cause adverse gastrointestinal effects. Two methods - high-resolution melting (HRM) and rhAmp® SNP genotyping - were developed to identify the ß-casein gene (CSN2) A1 and A2 alleles directly in milk. DNA milk samples from 45 animals were examined and 10 samples were also sequenced to confirm the accuracy of the assays. The analytical sensitivities of both strategies for A1 allele identification were evaluated by testing decreasing dilutions of A1 allele DNA copies (500 - 5 copies) in the A2 sample. The limits of detection for A1 in A2 samples were 10% (100 copies) and 2% (10 copies) for HRM and rhAmp, respectively. Both techniques were specific, differentiating between A1 and A2 alleles. However, we recommend rhAmp genotyping testing over HRM because of its enhanced sensitivity for A1.

Novel human sex-typing strategies based on the autism candidate gene NLGN4X and its male-specific gametologue NLGN4Y.

BACKGROUND: Since the early days of PCR techniques, sex identification, "sex-typing," of genomic DNA samples has been a fundamental part of human forensic analysis but also in animal genetics aiming at strategic livestock breeding. Most analyses are employing the AMELX/AMELY gene loci on the X and Y chromosomes present in most mammals. We hypothesize that sex-typing in humans is also possible based on the genes NLGN4X and NLGN4Y, which represent X and Y chromosome-specific copies of a common ancestral neuroligin-4 orthologue. METHODS: Genomic DNA was isolated from human blood and buccal cell samples (total n = 111) and submitted to two different strategies: (a) a traditional two-primer PCR approach detecting an insertion/deletion (indel) polymorphism immediately upstream of the translational start on exon 1 and (b) detection of a single nucleotide polymorphism, SNP, on the translational stop carrying exon 7. The SNP detection was based on a quantitative PCR approach (rhAMP genotyping) employing DNA/RNA hybrid oligonucleotides that were blocked and which could only be activated upon perfect annealing to the target DNA sequence. RESULTS: All indel PCR-tested human DNA samples showed two bands for males representing X- and Y-specific copies of NLGN4 and a single band for female samples, i.e., homozygosity of NLGN4X and absence of NLGN4Y, in accordance with the self-reported sex of the donors. These results were in perfect agreement with the results of the rhAMP-based SNP-detection method: all males were consequently positive for both alleles, representing either SNP variant, and females were interpreted as homozygous regarding the SNP variant found in NLGN4X. Both methods have shown reliable and consistent results that enabled us to infer the sex of donor DNA samples across different ethnicities. CONCLUSIONS: These results indicate that the detection of human NLGN4X/Y is a suitable alternative to previously reported methods employing gene loci such as AMELX/Y. Furthermore, this is the first report applying successfully the rhAMP-genotyping strategy as a means for SNP-based sex-typing, which consequently will be applicable to other gene loci or different species as well.

Comparison of three PCR-based assays for SNP genotyping in plants.

BACKGROUND: PCR allelic discrimination technologies have broad applications in the detection of single nucleotide polymorphisms (SNPs) in genetics and genomics. The use of fluorescence-tagged probes is the leading method for targeted SNP detection, but assay costs and error rates could be improved to increase genotyping efficiency. A new assay, rhAmp, based on RNase H2-dependent PCR (rhPCR) combined with a universal reporter system attempts to reduce error rates from primer/primer and primer/probe dimers while lowering costs compared to existing technologies. Before rhAmp can be widely adopted, more experimentation is required to validate its effectiveness versus established methods. RESULTS: The aim of this study was to compare the accuracy, sensitivity and costs of TaqMan, KASP, and rhAmp SNP genotyping methods in sugar beet (Beta vulgaris L.). For each approach, assays were designed to genotype 33 SNPs in a set of 96 sugar beet individuals obtained from 12 parental lines. The assay sensitivity was tested using a series of dilutions from 100 to 0.1 ng per PCR reaction. PCR was carried out on the QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific, USA). The call-rate, defined as the percentage of genotype calls relative to the possible number of calls, was 97.0, 97.6, and 98.1% for TaqMan, KASP, and rhAmp, respectively. For rhAmp SNP, 24 of the 33 SNPs demonstrated 100% concordance with other two technologies. The genotype concordance with either technologies for the other 9 targets was above 99% (99.34-99.89%). CONCLUSION: The sensitivity test demonstrated that TaqMan and rhAmp were able to successfully determine SNP genotypes using as little as 0.2 ng DNA per reaction, while the KASP was unable to ascertain SNP states below 0.9 ng of DNA per reaction. Comparative cost per reaction was also analyzed with rhAmp SNP offering the lowest cost per reaction. In conclusion, rhAmp produced more calls than either TaqMan or KASP, higher signal to NTC data while offering the lowest cost per reaction.