Allele-specific PCR with fluorescently labeled probes: criteria for selecting primers for genotyping.

Single-nucleotide polymorphisms (SNPs) can serve as reliable markers in genetic engineering, selection, screening examinations, and other fields of science, medicine, and manufacturing. Whole-genome sequencing and genotyping by sequencing can detect SNPs with high specificity and identify novel variants. Nonetheless, in situations where the interest of researchers is individual specific loci, these methods become redundant, and their cost, the proportion of false positive and false negative results, and labor costs for sample preparation and analysis do not justify their use. Accordingly, accurate and rapid methods for genotyping individual alleles are still in demand, especially for verification of candidate polymorphisms in analyses of association with a given phenotype. One of these techniques is genotyping using TaqMan allele-specific probes (TaqMan dual labeled probes). The method consists of real-time PCR with a pair of primers and two oligonucleotide probes that are complementary to a sequence near a given locus in such a way that one probe is complementary to the wild-type allele, and the other to a mutant one. Advantages of this approach are its specificity, sensitivity, low cost, and quick results. It makes it possible to distinguish alleles in a genome with high accuracy without additional manipulations with DNA samples or PCR products; hence the popularity of this method in genetic association studies in molecular genetics and medicine. Due to advancements in technologies for the synthesis of oligonucleotides and improvements in techniques for designing primers and probes, we can expect expansion of the possibilities of this approach in terms of the diagnosis of hereditary diseases. In this article, we discuss in detail basic principles of the method, the processes that influence the result of genotyping, criteria for selecting optimal primers and probes, and the use of locked nucleic acid modifications in oligonucleotides as well as provide a protocol for the selection of primers and probes and for PCR by means of rs11121704 as an example. We hope that the presented protocol will allow research groups to independently design their own effective assays for testing for polymorphisms of interest.

Dirus complex species identification PCR (DiCSIP) improves the identification of Anopheles dirus complex from the Greater Mekong Subregion.

BACKGROUND: The Anopheles dirus complex plays a significant role as a malaria vector in the Greater Mekong Subregion (GMS), with varying degrees of vector competence among species. Accurate identification of sibling species in this complex is essential for understanding malaria transmission dynamics and deploying effective vector control measures. However, the original molecular identification assay, Dirus allele-specific polymerase chain reaction (AS-PCR), targeting the ITS2 region, has pronounced nonspecific amplifications leading to ambiguous results and misidentification of the sibling species. This study investigates the underlying causes of these inconsistencies and develops new primers to accurately identify species within the Anopheles dirus complex. METHODS: The AS-PCR reaction and thermal cycling conditions were modified to improve specificity for An. dirus member species identification. In silico analyses with Benchling and Primer-BLAST were conducted to identify problematic primers and design a new set for Dirus complex species identification PCR (DiCSIP). DiCSIP was then validated with laboratory and field samples of the An. dirus complex. RESULTS: Despite several optimizations by reducing primer concentration, decreasing thermal cycling time, and increasing annealing temperature, the Dirus AS-PCR continued to produce inaccurate identifications for Anopheles dirus, Anopheles scanloni, and Anopheles nemophilous. Subsequently, in silico analyses pinpointed problematic primers with high Guanine-Cytosine (GC) content and multiple off-target binding sites. Through a series of in silico analyses and laboratory validation, a new set of primers for Dirus complex species identification PCR (DiCSIP) has been developed. DiCSIP primers improve specificity, operational range, and sensitivity to identify five complex member species in the GMS accurately. Validation with laboratory and field An. dirus complex specimens demonstrated that DiCSIP could correctly identify all samples while the original Dirus AS-PCR misidentified An. dirus as other species when used with different thermocyclers. CONCLUSIONS: The DiCSIP assay offers a significant improvement in An. dirus complex identification, addressing challenges in specificity and efficiency of the previous ITS2-based assay. This new primer set provides a valuable tool for accurate entomological surveys, supporting effective vector control strategies to reduce transmission and prevent malaria re-introducing in the GMS.

Genetic Diversity and Genome-Wide Association Analysis of the Hulled/Naked Trait in a Barley Collection from Shanghai Agricultural Gene Bank.

Barley is one of the most important cereal crops in the world, and its value as a food is constantly being revealed, so the research into and the use of barley germplasm are very important for global food security. Although a large number of barley germplasm samples have been collected globally, their specific genetic compositions are not well understood, and in many cases their origins are even disputed. In this study, 183 barley germplasm samples from the Shanghai Agricultural Gene Bank were genotyped using genotyping-by-sequencing (GBS) technology, SNPs were identified and their genetic parameters were estimated, principal component analysis (PCA) was preformed, and the phylogenetic tree and population structure of the samples were also analyzed. In addition, a genome-wide association study (GWAS) was carried out for the hulled/naked grain trait, and a KASP marker was developed using an associated SNP. The results showed that a total of 181,906 SNPs were identified, and these barley germplasm samples could be roughly divided into three categories according to the phylogenetic analysis, which was generally consistent with the classification of the traits of row type and hulled/naked grain. Population structure analysis showed that the whole barley population could be divided into four sub-populations (SPs), the main difference from previous classifications being that the two-rowed and the hulled genotypes were sub-divided into two SPs. The GWAS analysis of the hulled/naked trait showed that many associated loci were unrelated to the Nud/nud locus, indicating that there might be new loci controlling the trait. A KASP marker was developed for one exon-type SNP on chromosome 7. Genotyping based on the KASP assay was consistent with that based on SNPs, indicating that the gene of this locus might be associated with the hulled/naked trait. The above work not only lays a good foundation for the future utilization of this barley germplasm population but it provides new loci and candidate genes for the hulled/naked trait.

Development of Novel KASP Markers for Improved Germination in Deep-Sown Direct Seeded Rice.

BACKGROUND: The lack of stable-high yielding and direct-seeded adapted varieties with better germination ability from deeper soil depth and availability of molecular markers are major limitation in achieving the maximum yield potential of rice under water and resource limited conditions. Development of high-throughput and trait-linked markers are of great interest in genomics-assisted breeding. The aim of present study was to develop and validate novel KASP (Kompetitive Allele-Specific PCR) markers associated with traits improving germination and seedling vigor of deep sown direct seeded rice (DSR). RESULTS: Out of 58 designed KASP assays, four KASP assays did not show any polymorphism in any of the eleven genetic backgrounds considered in the present study. The 54 polymorphic KASP assays were then validated for their robustness and reliability on the F1s plants developed from eight different crosses considered in the present study. The third next validation was carried out on 256 F3:F4 and 713 BC3F2:3 progenies. Finally, the reliability of the KASP assays was accessed on a set of random 50 samples from F3:F4 and 80-100 samples from BC3F2:3 progenies using the 10 random markers. From the 54 polymorphic KASP, based on the false positive rate, false negative rate, KASP utility in different genetic backgrounds and significant differences in the phenotypic values of the positive (desirable) and negative (undesirable) traits, a total of 12 KASP assays have been selected. These 12 KASP include 5 KASP on chromosome 3, 1 on chromosome 4, 3 on chromosome 7 and 3 on chromosome 8. The two SNPs lying in the exon regions of LOC_Os04g34290 and LOC_Os08g32100 led to non-synonymous mutations indicating a possible deleterious effect of the SNP variants on the protein structure. CONCLUSION: The present research work will provide trait-linked KASP assays, improved breeding material possessing favourable alleles and breeding material in form of expected pre-direct-seeded adapted rice varieties. The marker can be utilized in introgression program during pyramiding of valuable QTLs/genes providing adaptation to rice under DSR. The functional studies of the genes LOC_Os04g34290 and LOC_Os08g32100 possessing two validated SNPs may provide valuable information about these genes.

Real-time PCR detection of PI*S and PI*Z alleles of SERPINA1 gene using SYBR green.

BACKGROUND: Alpha-1 antitrypsin deficiency is an underdiagnosed genetic condition that predisposes to pulmonary complications and is mainly caused by rs28929474 (PI*Z allele) and rs17580 (PI*S allele) mutations in the SERPINA1 gene. OBJECTIVE: Development of a homogeneous genotyping test for detection of PI*S and PI*Z alleles based on the principles of allele-specific PCR and amplicon melting analysis with a fluorescent dye. METHODS: Sixty individuals, which included all possible genotypes that result from combinations of rs28929474 and rs17580 single nucleotide variants, were assayed with tailed allele-specific primers and SYBR Green dye in a real-time PCR machine. RESULTS: A clear discrimination of mutant and wild-type variants was achieved in the genetic loci that define PI*S and PI*Z alleles. Specific amplicons showed a difference of 2.0 °C in melting temperature for non-S and S variants and of 2.9 °C for non-Z and Z variants. CONCLUSIONS: The developed genotyping method is robust, fast, and easily scalable on a standard real-time PCR platform. While it overcomes the handicaps of non-homogeneous approaches, it greatly reduces genotyping costs compared with other homogeneous approaches.

Characterization of a New Citrus Mutant Induced by Gamma Irradiation with a Unique Fruit Shape, Gwonje-Early, and Determination of Specific Selection Markers Using Allele-Specific PCR.

Gamma-ray irradiation is one of the most widely used mutagens worldwide. We previously conducted mutation breeding using gamma irradiation to develop new Citrus unshiu varieties. Among these mutants, Gwonje-early had an ovate shape, a protrusion of the upper part of the fruit, and a large fruit size compared with wild-type (WT) fruits. We investigated the external/internal morphological characteristics and fruit sugar/acid content of Gwonje-early. Additionally, we investigated genome-wide single-nucleotide polymorphisms (SNPs) and insertion/deletion (InDel) variants in Gwonje-early using whole-genome re-sequencing. Functional annotation by Gene Ontology analysis confirmed that InDels were more commonly annotated than SNPs. To identify specific molecular markers for Gwonje-early, allele-specific PCR was performed using homozygous SNPs detected via Gwonje-early genome re-sequencing. The GJ-SNP1 and GJ-SNP4 primer sets were effectively able to distinguish Gwonje-early from the WT and other commercial citrus varieties, demonstrating their use as specific molecular markers for Gwonje-early. These findings also have important implications in terms of intellectual property rights and the variety protection of Gwonje-early. Our results may provide insights into the understanding of morphological traits and the molecular breeding mechanisms of citrus species.

KASP: a high-throughput genotyping system and its applications in major crop plants for biotic and abiotic stress tolerance.

Advances in plant molecular breeding have resulted in the development of new varieties with superior traits, thus improving the crop germplasm. Breeders can screen a large number of accessions without rigorous and time-consuming phenotyping by marker-assisted selection (MAS). Molecular markers are one of the most imperative tools in plant breeding programmes for MAS to develop new cultivars possessing multiple superior traits. Single nucleotide polymorphisms (SNPs) are ideal for MAS due to their low cost, low genotyping error rates, and reproducibility. Kompetitive Allele Specific PCR (KASP) is a globally recognized technology for SNP genotyping. KASP is an allele-specific oligo extension-based PCR assay that uses fluorescence resonance energy transfer (FRET) to detect genetic variations such as SNPs and insertions/deletions (InDels) at a specific locus. Additionally, KASP allows greater flexibility in assay design, which leads to a higher success rate and the capability to genotype a large population. Its versatility and ease of use make it a valuable tool in various fields, including genetics, agriculture, and medical research. KASP has been extensively used in various plant-breeding applications, such as the identification of germplasm resources, quality control (QC) analysis, allele mining, linkage mapping, quantitative trait locus (QTL) mapping, genetic map construction, trait-specific marker development, and MAS. This review provides an overview of the KASP assay and emphasizes its validation in crop improvement related to various biotic and abiotic stress tolerance and quality traits.

Genome-wide association study and development of molecular markers for yield and quality traits in peanut (Arachis hypogaea L.).

BACKGROUND: This study aims to decipher the genetic basis governing yield components and quality attributes of peanuts, a critical aspect for advancing molecular breeding techniques. Integrating genotype re-sequencing and phenotypic evaluations of seven yield components and two grain quality traits across four distinct environments allowed for the execution of a genome-wide association study (GWAS). RESULTS: The nine phenotypic traits were all continuous and followed a normal distribution. The broad heritability ranged from 88.09 to 98.08%, and the genotype-environment interaction effects were all significant. There was a highly significant negative correlation between protein content (PC) and oil content (OC). The 10× genome re-sequencing of 199 peanut accessions yielded a total of 631,988 high-quality single nucleotide polymorphisms (SNPs), with 374 significant SNP loci identified in association with the nine traits of interest. Notably, 66 of these pertinent SNPs were detected in multiple environments, and 48 of them were linked to multiple traits of interest. Five loci situated on chromosome 16 (Chr16) exhibited pleiotropic effects on yield traits, accounting for 17.64-32.61% of the observed phenotypic variation. Two loci on Chr08 were found to be strongly associated with protein and oil contents, accounting for 12.86% and 14.06% of their respective phenotypic variations, respectively. Linkage disequilibrium (LD) block analysis of these seven loci unraveled five nonsynonymous variants, leading to the identification of one yield-related candidate gene and two quality-related candidate genes. The correlation between phenotypic variation and SNP loci in these candidate genes was validated by Kompetitive allele-specific PCR (KASP) marker analysis. CONCLUSIONS: Overall, molecular markers were developed for genetic loci associated with yield and quality traits through a GWAS investigation of 199 peanut accessions across four distinct environments. These molecular tools can aid in the development of desirable peanut germplasm with an equilibrium of yield and quality through marker-assisted breeding.

Genetic association of novel SNPs in HK-1 (rs201626997) and HK-3 (rs143604141) with type 2 diabetes mellitus in Bangladeshi population.

BACKGROUND: Hexokinase, a key enzyme in glycolysis, has isoforms like HK-1, HK-2, HK-3, and Glucokinase. Unpublished exome sequencing data showed that two novel polymorphisms in HK-1 rs201626997 (G/T) and HK-3 rs143604141 (G/A) exist in the Bangladeshi population. We investigated the possible relationship of these SNPs with T2DM. MATERIALS AND METHODS: Peripheral blood samples from the study participants were used to isolate their genomic DNA. An allele-specific PCR was standardized that can discriminate between the wild-type and mutant-type alleles of HK-1 (rs201626997) and HK-3 (rs143604141) polymorphisms. The data was analyzed by SPSS for statistics. RESULTS: We performed allele-specific PCR for 249 diabetic patients and 195 control samples. For HK-1 (rs201626997), 24 (5.4%) have a mutant allele, and for HK-3 (rs143604141), 25 (5.6%) are mutant. There is no significant relationship between the individuals' disease condition and the HK-1 polymorphism (P value 0.537). But the GA genotype of the HK-3 rs143604141 pertains to an increased risk of diabetes (P value 0.039). HK-3 rs143604141 polymorphism has a moderate correlation (P value 0.078, OR, 3.11, 95% CI, 0.88-10.94) with a family diabetic history. Both polymorphisms showed no significant correlation with gender or BMI. However, hexokinase-1 polymorphism significantly related with diastolic blood pressure (P value 0.048). CONCLUSION: This study will help us to easily detect the polymorphisms of HK-1 (rs201626997) and HK-3 (rs143604141) in different populations of the world. Further studies with a greater number of participants and more physiological information are required to better understand the underlying genetic causes of T2DM susceptibility in Bangladesh.

Rapid and Reliable HLA-B*59:01 Genotyping to Prevent Carbonic Anhydrase Inhibitor-Induced Severe Cutaneous Adverse Reactions.

OBJECTIVE: Carbonic anhydrase inhibitors (CAIs) are intraocular pressure-reducing medications used in ophthalmology. Human leukocyte antigen-B*59:01 (HLA-B*59:01) is strongly associated with CAI-induced severe cutaneous adverse reactions (SCARs). This study aimed to develop and validate a rapid and economical screening method for HLA-B*59:01 to prevent carbonic anhydrase inhibitor-induced SCARs. METHODS: Duplex allele-specific polymerase chain reaction (PCR) with an internal control was performed for HLA-B*59:01 genotyping. The accuracy of duplex allele-specific PCR for HLA-B*59:01 genotyping was evaluated in 200 blood samples, using sequence-based typing (SBT) as the reference method. RESULTS: In total, 50 HLA-B*59:01-positive and 150 HLA-B*59:01-negative results obtained using duplex allele-specific PCR were in complete agreement with the SBT results. CONCLUSION: Duplex allele-specific PCR is a rapid, reliable, and economical assay for screening the HLA-B*59:01 allele.

Development of an allele-specific PCR (AS-PCR) method for identifying high-methyl eugenol-containing purple Tulsi (Ocimum tenuiflorum L.) in market samples.

BACKGROUND: Ocimum tenuiflorum L. is a highly traded medicinal with several therapeutic values. Green Tulsi and purple Tulsi are two subtypes in O. tenuiflorum and both have the same medicinal properties. Recent reports have revealed that purple Tulsi contains higher quantities of methyl eugenol (ME), which is moderately toxic and potentially carcinogenic. Therefore, we developed an allele-specific PCR (AS-PCR) method to distinguish the green and purple Tulsi. METHODS AND RESULT: Using the green Tulsi as a reference, 12 single nucleotide polymorphisms (SNPs) and 10 insertions/deletions (InDels) were identified in the chloroplast genome of the purple Tulsi. The C > T SNP at the 1,26,029 position in the ycf1 gene was selected for the development of the AS-PCR method. The primers were designed to amplify 521 bp and 291 bp fragments specific to green and purple Tulsi, respectively. This AS-PCR method was validated in 10 accessions from each subtype and subsequently verified using Sanger sequencing. Subsequently, 30 Tulsi powder samples collected from the market were subjected to molecular identification by AS-PCR. The results showed that 80% of the samples were purple Tulsi, and only 3.5% were green Tulsi. About 10% of the samples were a mixture of both green and purple Tulsi. Two samples (6.5%) did not contain O. tenuiflorum and were identified as O. gratissimum. CONCLUSION: The market samples of Tulsi were predominantly derived from purple Tulsi. The AS-PCR method will be helpful for quality control and market surveillance of Tulsi herbal powders.

Genetic Association of Transcription Factor 7-Like-2 rs7903146 Polymorphism With Type 2 Diabetes Mellitus.

INTRODUCTION: Type 2 diabetes mellitus (T2DM) mainly results from the inability of muscle, fat, and liver cells to uptake glucose due to insulin resistance or deficiency of insulin production by the pancreas. Predisposition to T2DM may be due to environmental, hereditary, or both factors. Although there are many genes involved in causing T2DM, transcription factor 7-like-2 gene (TCF7L2) rs7903146 (C/T) single nucleotide polymorphism (SNP) found in genome-wide association studies (GWAS) is susceptible to T2DM. TCF7L2 is involved in pancreatic beta cell proliferation and differentiation via the Wnt signaling mechanism. OBJECTIVES: To find the genetic association of TCF7L2 rs7903146 (C/T) gene polymorphism in patients with T2DM. METHODS: A case-control study was conducted on 194 T2DM patients recruited from the endocrinology department at Indira Gandhi Institute of Medical Sciences, Patna, and 180 non-diabetic healthy controls that were age and sex-matched with the patients. All clinical examination and biochemical investigations like glycosylated hemoglobin (HbA1c), total cholesterol, triglycerides, high-density lipoprotein-cholesterol, and low-density lipoprotein-cholesterol; and determination of TCF7L2 gene polymorphism by allele-specific polymerase chain reaction (AS-PCR) were carried out for each subject. RESULTS:  The T allele of the rs7903146 (C/T) SNP was associated with a two-fold higher risk of T2DM and the heterozygous genotype (CT) with a 1.96 times higher risk. CONCLUSION: There is a high association of this SNP with the development of T2DM in the eastern Indian population. Serial monitoring of HbA1c should be done in an individual having this type of polymorphism for early detection of T2DM to prevent future complications.

Phosphoramidate Azole Oligonucleotides for Single Nucleotide Polymorphism Detection by PCR.

Detection of the Kirsten rat sarcoma gene (KRAS) mutational status is an important factor for the treatment of various malignancies. The most common KRAS-activating mutations are caused by single-nucleotide mutations, which are usually determined by using PCR, using allele-specific DNA primers. Oligonucleotide primers with uncharged or partially charged internucleotide phosphate modification have proved their ability to increase the sensitivity and specificity of various single nucleotide mutation detection. To enhance the specificity of single nucleotide mutation detection, the novel oligonucleotides with four types of uncharged and partially charged internucleotide phosphates modification, phosphoramide benzoazole (PABA) oligonucleotides (PABAO), was used to prove the concept on the KRAS mutation model. The molecular effects of different types of site-specific PABA modification in a primer or a template on a synthesis of full-length elongation product and PCR efficiency were evaluated. The allele-specific PCR (AS-PCR) on plasmid templates showed a significant increase in analysis specificity without changes in Cq values compared with unmodified primer. PABA modification is a universal mismatch-like disturbance, which can be used for single nucleotide polymorphism discrimination for various applications. The molecular insights of the PABA site-specific modification in a primer and a template affect PCR, structural features of four types of PABAO in connection with AS-PCR results, and improvements of AS-PCR specificity support the further design of novel PCR platforms for various biological targets testing.

Identification of two major QTLs for pod shell thickness in peanut (Arachis hypogaea L.) using BSA-seq analysis.

BACKGROUND: Pod shell thickness (PST) is an important agronomic trait of peanut because it affects the ability of shells to resist pest infestations and pathogen attacks, while also influencing the peanut shelling process. However, very few studies have explored the genetic basis of PST. RESULTS: An F2 segregating population derived from a cross between the thick-shelled cultivar Yueyou 18 (YY18) and the thin-shelled cultivar Weihua 8 (WH8) was used to identify the quantitative trait loci (QTLs) for PST. On the basis of a bulked segregant analysis sequencing (BSA-seq), four QTLs were preliminarily mapped to chromosomes 3, 8, 13, and 18. Using the genome resequencing data of YY18 and WH8, 22 kompetitive allele-specific PCR (KASP) markers were designed for the genotyping of the F2 population. Two major QTLs (qPSTA08 and qPSTA18) were identified and finely mapped, with qPSTA08 detected on chromosome 8 (0.69-Mb physical genomic region) and qPSTA18 detected on chromosome 18 (0.15-Mb physical genomic region). Moreover, qPSTA08 and qPSTA18 explained 31.1-32.3% and 16.7-16.8% of the phenotypic variation, respectively. Fifteen genes were detected in the two candidate regions, including three genes with nonsynonymous mutations in the exon region. Two molecular markers (Tif2_A08_31713024 and Tif2_A18_7198124) that were developed for the two major QTL regions effectively distinguished between thick-shelled and thin-shelled materials. Subsequently, the two markers were validated in four F2:3 lines selected. CONCLUSIONS: The QTLs identified and molecular markers developed in this study may lay the foundation for breeding cultivars with a shell thickness suitable for mechanized peanut shelling.

An idea to explore: Determination of single nucleotide polymorphisms in alcohol metabolism-related genes using PCR-based assays to understand the link between an individual's genotype and phenotype.

Here, we propose a laboratory exercise to quickly determine single nucleotide polymorphisms (SNPs) in human alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) genes involved in alcohol metabolism. In this exercise, two different genotyping methods based on polymerase chain reaction (PCR), namely allele-specific (AS) PCR and a PCR-restriction fragment polymorphism (RFLP) analysis, can be performed under the same PCR program (2-step × 35 cycles, 35 min total) in parallel using a hair root lysate as a template. In AS-PCR, the target regions of the G- or A-alleles of both genes are allele-specifically amplified in a single PCR tube. In the PCR-RFLP analysis, the two genes are amplified simultaneously in a single tube, and then a portion of the PCR product is double-digested with restriction enzymes MslI and Eam1104I for 5 min. The resulting reaction products of each method are electrophoresed side by side, and the genotypes are determined from the DNA band patterns. With the optimized protocol, the whole process from template preparation to genotyping can be completed in about 75 min. During PCR, students also perform an ethanol patch test to estimate their ability to metabolize alcohol. This series of experiments can help students learn the principles and applications of PCR/SNP analyses. By comparing the genotypes revealed by PCR and the phenotypes revealed by the patch tests, students can gain a better understanding of the clinical value of genetic testing.

Performance characteristics of Allele-Specific PCR (ASPCR) in detecting drug resistance mutations among non-B HIV-1 Variants.

Allele-Specific Polymerase Chain Reaction (ASPCR) is an affordable point-mutation assay whose validation could improve the detection of HIV-1 drug resistance mutations (DRMs) in resource-limited settings (RLS). We assessed the performance of ASPCR onforty-four non-B HIV-1 plasma samples from patients who were ARV treated in failure in N'Djamena-Chad. Viral RNA was reverse-transcribed and amplified using LightCycler® FastStart DNA MasterPLUS SYBR Green I. Detection of six major DRMs (K70R, K103N, Y181C, M184V, T215F, T215Y) was evaluated on Roche LightCycler®480 automated system (with dilutions 0.01-100%). ASPCR-results were compared to Sanger-sequencing (gold-standard). Correlations of mutation curves were excellent (R2 >0.97); all DRMs were detected with desirable mutant/wild-type threshold differences (ΔCt≥9) except K70R(ΔCtK70R=6; ΔCtK103N=13; ΔCtM184V=9; ΔCtT215F=12; ΔCtT215Y=12; ΔCtY181C=9) and positive controls were below required thresholds. Also, ASPCR reproducibility on DRMs was assessed by using dilutions of intra-assay and inter-assay coefficient of variations respectively with a threshold of less than 50(i.e.<0.50 variation) which are;: K70R (0.02-0.28 vs. 0.12-0.37), K103N (0.08-0.42 vs. 0.12-0.37), Y181C (0.12-0.39 vs. 0.31-0.37), M184V (0.13-0.39 vs. 0.23-0.42), T215F (0.05-0.43 vs. 0.04-0.45) and T215Y (0.13-0.41 vs. 0.19-0.41). DRM detection-rate by ASPCR vs Sanger was respectively: M184V (63.6% vs. 38.6%); T215F (18.1% vs. 9.1%); T215Y (6.8% vs. 2.3%); K70R (4.5% vs. 2.3%). K103N (22.7% vs. 13.6%); Y181C (13.6% vs. 11.4%). Correlations of mutation curves were excellent (R2 >0.97); all DRMs were detected with desirable mutant/wild-type threshold differences (ΔCt≥9) except K70R(ΔCtK70R=6; ΔCtK103N=13; ΔCtM184V=9; ΔCtT215F=12; ΔCtT215Y=12; ΔCtY181C=9) and positive controls were below required thresholds. Also, ASPCR reproducibility on DRMs was assessed by using dilutions of intra-assay and inter-assay coefficient of variations respectively with a threshold of less than 50(i.e.<0.50 variation) which are;: K70R (0.02-0.28 vs. 0.12-0.37), K103N (0.08-0.42 vs. 0.12-0.37), Y181C (0.12-0.39 vs. 0.31-0.37), M184V (0.13-0.39 vs. 0.23-0.42), T215F (0.05-0.43 vs. 0.04-0.45) and T215Y (0.13-0.41 vs. 0.19-0.41). DRM detection-rate by ASPCR vs Sanger was respectively: M184V (63.6% vs. 38.6%); T215F (18.1% vs. 9.1%); T215Y (6.8% vs. 2.3%); K70R (4.5% vs. 2.3%). K103N (22.7% vs. 13.6%); Y181C (13.6% vs. 11.4%). ASPCR appears more efficient for detecting DRMs on diverse HIV-1 non-B circulating in RLS like Chad.

Using association mapping and local interval haplotype association analysis to improve the cotton drought stress response.

Drought stress has a serious impact on the growth and development of cotton. To explore the relevant molecular mechanism of the drought stress response in cotton, gene mapping based on the QTL interval mapped by simplified genome BSA-seq of the drought-resistance-related RIL population was performed. A QTL region spanning 2.02 Mb on chromosome D07 was selected, and 201 resource materials were genotyped using 9 KASP markers in the interval. After local interval haplotype association analysis, the overlap of the 110 kb peak region confirmed the reliability of this region, and at the same time, the role of GhGF14-30, the only gene in the overlapping region, was modeled in the response of cotton to drought stress. qRTPCR analysis of the materials and population parents proved that this gene plays a role in the drought stress response in cotton. Virus-induced gene silencing proved the importance of this gene in drought-sensitive materials, and drought-resistance-related marker genes also proved that the GhGF14-30 gene may play an important role in the ABA and SOS signaling pathways. This study provides a basis for mining drought stress response functional genes in cotton and lays the foundation for the molecular mechanism of the GhGF14-30 gene in response to drought stress in cotton.

Incidence and prognostic impact of U2AF1 mutations and other gene alterations in myelodysplastic neoplasms with isolated 20q deletion.

BACKGROUND: In myelodysplastic neoplasms (MDS), the 20q deletion [del(20q)] is a recurrent chromosomal abnormality that it has a high co-occurrence with U2AF1 mutations. Nevertheless, the prognostic impact of U2AF1 in these MDS patients is uncertain and the possible clinical and/or prognostic differences between the mutation type and the mutational burden are also unknown. METHODS: Our study analyzes different molecular variables in 100 MDS patients with isolated del(20q). RESULTS & CONCLUSIONS: We describe the high incidence and negative prognostic impact of U2AF1 mutations and other alterations such as in ASXL1 gene to identify prognostic markers that would benefit patients to receive earlier treatment.

Effectiveness of TaDreb-B1 and 1-FEH w3 KASP Markers in Spring and Winter Wheat Populations for Marker-Assisted Selection to Improve Drought Tolerance.

Due to the advances in DNA markers, kompetitive allele-specific PCR (KASP) markers could accelerate breeding programs and genetically improve drought tolerance. Two previously reported KASP markers, TaDreb-B1 and 1-FEH w3, were investigated in this study for the marker-assisted selection (MAS) of drought tolerance. Two highly diverse spring and winter wheat populations were genotyped using these two KASP markers. The same populations were evaluated for drought tolerance at seedling (drought stress) and reproductive (normal and drought stress) growth stages. The single-marker analysis revealed a high significant association between the target allele of 1-FEH w3 and drought susceptibility in the spring population, while the marker-trait association was not significant in the winter population. The TaDreb-B1 marker did not have any highly significant association with seedling traits, except the sum of leaf wilting in the spring population. For field experiments, SMA revealed very few negative and significant associations between the target allele of the two markers and yield traits under both conditions. The results of this study revealed that the use of TaDreb-B1 provided better consistency in improving drought tolerance than 1-FEH w3.

Differentiation of Andean and Mesoamerican accessions in a proposed core collection of grain amaranths.

Grain amaranths are made up of three New World species of pseudo-cereals with C4 photosynthesis from the dicotyledonous family Amaranthaceae and the genus Amaranthus. They originate in two ecoregions of the Americas, namely, the inter-Andean valleys of South America and the volcanic axis and lowlands of Mexico and Central America. These correspond to two centers of domestications for Andean and Mesoamerican crops, with one cultivated species found in the first region and two found in the latter region. To date, no core collection has been made for the grain amaranths in the United States Department of Agriculture (USDA) germplasm system. In this study, our objective was to create a core for the 2,899 gene bank accessions with collection site data by town or farm site of which 1,090 have current geo-referencing of latitude and longitude coordinates. We constituted the core with 260 genotypes of Amaranthus, which we evaluated with 90 single-nucleotide polymorphism markers. Our goal was to distinguish between Andean and Mesoamerican gene pools of amaranths, including the cultivated species and three possible progenitor or wild relative ancestors along with two more species in an outgroup. Population structure, clustering, and discriminant analysis for principal components showed that Andean species Amaranthus caudatus and Amaranthus quitensis shared fewer alleles with Mesoamerican species Amaranthus cruentus and Amaranthus hypochondriacus, compared to each group individually. Amaranthus hybridus was a bridge species that shared alleles with both regions. Molecular markers have the advantage over morphological traits at quickly distinguishing the Andean and Mesoamerican cultivars and have the added benefit of being useful for following inter-species crosses and introgression.