Allele-specific PCR Assay Research Articles

Development and Utilization of Functional Kompetitive Allele-Specific PCR Markers for Key Genes Underpinning Fiber Length and Strength in Gossypium hirsutum L.

Fiber length (FL) and fiber strength (FS) are the important indicators of fiber quality in cotton. Longer and stronger fibers are preferred for manufacturing finer yarns in the textile industry. Functional markers (FMs) designed from polymorphic sites within gene sequences attributing to phenotypic variation are highly efficient when used for marker-assisted selection (MAS) in breeding superior varieties with longer FL and higher FS. The aims of this study were to develop FMs via kompetitive allele-specific PCR (KASP) assays and to validate the efficacy of the FMs for allele discrimination and the potential value in practice application. We used four single-nucleotide polymorphism markers and 360 cotton accessions and found that two FMs, namely, D11_24030087 and A07_72204443, could effectively differentiate accessions of different genotypes with higher consistency to phenotype. The appeared frequencies of varieties harbored Hap2 (elite alleles G and T) with longer FL (> the mean of accessions with non-elite allele, 28.50 mm) and higher FS (> the mean of accessions with non-elite allele, 29.06 cN•tex–1) were 100 and 72.7%, respectively, which was higher than that of varieties harbored only on a single elite allele (G or T, 77.9 or 61.9%), suggesting a favorable haplotype for selecting varieties with superior FL and FS. These FMs could be valuable for the high-throughput selection of superior materials by providing genotypic information in cotton breeding programs.

Molecular sexual determinants in Pistacia genus by KASP assay.

Pistacia is a genus of dioecious plant species whose trees can take 4-5years to reach the economically valuable fruit-bearing stage. The fruits have great importance as raw material in the food, healthcare, and baking industries. For that reason, the identification of individual plants in the early juvenile period for the pollination and positioning of trees is crucial for growers. The objective of this study is to develop markers for each Pistacia species that can help in screening the sex of plant seedlings before they reach the reproductive stage, without waiting for morphological characteristics to appear. Within this context, by using the power of the kompetitive allele-specific PCR (KASP) assay technology as a marker screening system, we successfully discriminated seven out of eight Pistacia species: P. atlantica, P. integerrima, P. khinjuk, P. mutica, P. terebinthus, P. vera, and P. lentiscus. We used a high-throughput DNA sequence read archive (SRA) to assemble a reference genome that was employed in our studies as a de novo bioinformatics method. Four genomic regions from SRA and three single-nucleotide polymorphism (SNP) positions from Kafkas et al. BMC Genomics 16:98, 2015) were selected and sequenced with collected plant material from predominantly the Antepfıstıgı Research Institute Collection Garden, and eight species were aligned intraspecifically for SNP mining. In total, 12 SNP markers were converted to KASP markers, and 5 of them (SNP-PIS-133396, SNP-PIS-167992, P-ATL-91951-565, P-INT-91951-256, P-KHI-91951-115) showed clear allelic discrimination between male and female plants. SNP-PIS-167992 and P-ATL-91951-565 were identified as the best marker assays because they showed allelic frequency differences for all individuals and for both homozygous and heterozygous characters. These markers could be the most comprehensive ones for the whole genus because they showed discriminative power for several species. This study is the first one to use the KASP assay for sex discrimination in Pistacia species, and it can be regarded as a precursor study for sex discrimination by KASP for plants in general.

Adult plant stem rust resistance in durum wheat Glossy Huguenot: mapping, marker development and validation.

Adult plant stem rust resistance locus, QSrGH.cs-2AL, was identified in durum wheat Glossy Huguenot and mendelised as Sr63. Markers closely linked with Sr63 were developed. An F3 population from a Glossy Huguenot (GH)/Bansi cross used in a previous Australian study was advanced to F6 for molecular mapping of adult plant stem rust resistance. Maturity differences among F6 lines confounded assessments of stem rust response. GH was crossed with a stem rust susceptible F6 recombinant inbred line (RIL), GHB14 (M14), with similar maturity and an F6:7 population was developed through single seed descent method. F7 and F8 RILs were tested along with the parents at different locations. The F6 individual plants and both parents were genotyped using the 90K single nucleotide polymorphism (SNP) wheat array. Stem rust resistance QTL on the long arms of chromosomes 1B (QSrGH.cs-1BL) and 2A (QSrGH.cs-2AL) were detected. QSrGH.cs-1BL and QSrGH.cs-2AL were both contributed by GH and explained 22% and 18% adult plant stem rust response variation, respectively, among GH/M14 RIL population. RILs carrying combinations of these QTL reduced more than 14% stem rust severity compared to those that possessed QSrGH.cs-1BL and QSrGH.cs-2AL individually. QSrGH.cs1BL was demonstrated to be the same as Sr58/Lr46/Yr29/Pm39 through marker genotyping. Lines lacking QSrGH.cs-1BL were used to Mendelise QSrGH.cs-2AL. Based on genomic locations of previously catalogued stem rust resistance genes and the QSrGH.cs-2AL map, it appeared to represent a new APR locus and was permanently named Sr63. SNP markers associated with Sr63 were converted to kompetetive allele-specific PCR (KASP) assays and were validated on a set of durum cultivars.

Map-Based Cloning and Characterization of Br-dyp1, a Gene Conferring Dark Yellow Petal Color Trait in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Flower color is an important trait in Brassica species. However, genes responsible for the dark yellow flower trait in Chinese cabbage have not been reported. In this study, we identified a dark-yellow-flowered Chinese cabbage line SD369. Genetic analysis indicated that the dark yellow flower trait in SD369 was controlled by a single recessive locus, Br-dyp1 (dark yellow petal color 1 in Brassica rapa). Using bulked segregant RNA sequencing and kompetitive allele-specific PCR assays, Br-dyp1 was fine-mapped to an interval of 53.6 kb on chromosome A09. Functional annotation analysis, expression analysis, and sequence variation analysis revealed that Bra037130 (BraA09.ZEP), which encodes a zeaxanthin epoxidase, was the most likely candidate gene for Br-dyp1. Carotenoid profile analysis suggested that Bra037130 (BraA09.ZEP) might participate in the epoxidation from zeaxanthin to violaxanthin. The 679 bp insertion in dark yellow petal caused premature stop codon, thus caused the loss-of-function of the enzyme zeaxanthin epoxidase (ZEP), which disturbed the carotenoid metabolism, and caused the increased accumulation of total carotenoid, and finally converted the flower color from yellow to dark yellow. Comparative transcriptome analysis also showed that the “carotenoid biosynthesis” pathway was significantly enriched, and genes involved in carotenoid degradation and abscisic acid biosynthesis and metabolism were significantly downregulated. Furthermore, we developed and validated the functional marker Br-dyp1-InDel for Br-dyp1. Overall, these results provide insight into the molecular basis of carotenoid-based flower coloration in B. rapa and reveal valuable information for marker-assisted selection breeding in Chinese cabbage.

Sexing assay for chickens and other birds for large-scale application based on a conserved sequence variant in CHD1 genes on W and Z chromosomes.

Several PCR-based methods are known for sexing of chickens and other birds (Çakmak et al., 2017; Eiras et al., 2018; Gruszczyńska & Grzegrzółka, 2021; Morinha et al., 2012). While some methods for bird sexing are suitable for large-scale analyses (Chen et al., 2012; Clinton et al., 2016; He et al., 2019; Margulis & Danielli, 2019; Morinha et al., 2013; Rosenthal et al., 2010), research is still ongoing because most of these methods are costly and time consuming. Here we report a newly developed, easy to use competitive allele-specific PCR (KASP) assay that is suitable for large-scale sexing in chickens and other birds. The KASP assay is based on an A/G difference in exon 17 between the W- and Z-chromosomal variants of the conserved chromodomain helicase DNA binding protein 1 (CHD1) in exon 17 (Figure S1). Sex-specific primers were designed up- and downstream of this variant in CHD1 genes to amplify a product of 46 bp (Figure 1; Table S1, Figure S1). The amplicon overlaps with the PCR product for sex genotyping of the method of Fridolfsson and Ellegren (1999) (Figure S1). Furthermore, similar sequences were obtained from NCBI databases using blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for duck, goose, quail and turkey and were aligned with the chicken sequence (Figure 1). In total, 734 chicken samples with known sex were analysed (Table S2). In addition, 55 samples of other species of the orders Galliformes and Anseriformes were analysed (Table S2). If the sex of non-chicken samples was unknown it was verified by multiplex PCR modified from Fridolfsson and Ellegren (1999) (Table S1, Figure S1). Samples were mainly taken from an extensive DNA collection, which was set up within the framework of the projects AVIANDIV (Lyimo et al., 2014) and SYNBREED (www.synbreed.tum.de). Each KASP reaction contained 20–50 ng of template DNA, KASP v. 4.0 2× Master mix standard ROX and the KASP-by-Design assay mix (LGC Genomics). The standard KASP thermal cycling conditions according to LGC protocols were performed in an Eppendorf Mastercycler (Eppendorf). After amplification, microplates were analysed with FLUOstar Omega (BMG Labtech) using excitation and emission values of 485/520 nm for the FAM-labelled-FRET cassette, 530/560 nm for the HEX-labelled-FRET cassette and 584/620 nm for the ROX standard. All chickens, ducks, geese, quails and turkeys were correctly assigned to their sex using the newly developed KASP assay (Table S2). This newly developed KASP assay is well suited to chicken, duck, goose, quail and turkey for efficient sex determination on a larger scale. The research leading to these results has received funding from the European Union's Horizon 2020 Research and Innovation Programme under the grant agreement no. 677353 IMAGE. The authors thank Alina Kirchhoff, Kim Köhler, Christin Moelders and Katrin Zimmermann for technical assistance. Open access funding was enabled and organised by ProjektDEAL. Open access funding enabled and organized by ProjektDEAL. The authors declare no potential conflicts of interest. The data supporting the findings of the present study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Development of Multiplex Allele-specific PCR Assay for BRAFV600E Mutation Detection in Human Cancer Cells

Development of Multiplex Allele-specific PCR Assay for BRAFV600E Mutation Detection in Human Cancer Cells

Selection for resistance to cassava mosaic disease in African cassava germplasm using single nucleotide polymorphism markers

Cassava mosaic disease (CMD) is one of the main constraints that hamper cassava production. Breeding for varieties that are CMD resistant is a major aim in cassava breeding programmes. However, the use of the conventional approach has its limitations, including a lengthy growth cycle and a low multiplication rate of planting materials. To increase breeding efficiency as well as genetic gain of traits, SNP markers can be used to screen and identify resistant genotypes. The objective of this study was to predict the performance of 145 cassava genotypes from open-pollinated crosses for CMD resistance using molecular markers. Two SNP markers (S12_7926132 and S14_4626854), previously converted into Kompetitive allele-specific PCR (KASP) assays, as well as CMD incidence and severity scores, were used for selection. About 76% of the genotypes were revealed to be resistant to CMD based on phenotypic scores, while over 24% of the total population were found to be susceptible. Significant effects were observed for alleles associated with marker S12_7926132 while the other marker had non-significant effects. The predictive accuracy (true positives and true negatives) of the major CMD2 locus on chromosome 12 was 77% in the population used in this study. Our study provides insight into the potential use of marker-assisted selection for CMD resistance in cassava breeding programmes.Significance: With an aim towards reducing the food insecurity rate in Africa, we report on the use of genetic tools for a fast and efficient release of new cassava varieties to benefit breeders, farmers and consumers, given the food and industrial importance of this staple crop. This study adds tremendous knowledge to phenotypic and molecular screening for CMD resistance. The outcome will encourage breeders in various cassava breeding programmes to accelerate genetic gains as well as increase breeding accuracy and efficiency for CMD resistance.

Validation and molecular analysis of β-1,3-GLU2 SNP marker associated with resistance to Colletotrichum gloeosporioides in mango (Mangifera indica L.)

Anthracnose caused by Colletotrichum gloeosporioides is one of the most destructive diseases of mango worldwide. Breeding for genetic resistance to anthracnose is necessary to improve the yield and fruit quality of mango. Here, we identified and analyzed the molecular basis of a SNP within the pathogenesis-related β-1,3-glucanase 2 (β-1,3-GLU2) gene putatively linked with resistance to anthracnose. This SNP designated ‘21881933’ causes a missense mutation (I196V) in the highly conserved glycosyl hydrolase family 17 (GH17) domain—a structural unit involved in physiologically important processes in plants, notably in response to biotic and abiotic stresses, activation of phytohormones, lignification, and cell wall remodeling. Structural analysis suggested that the I196V mutation results in unfavorable conformational changes in the enzyme's (β/α)8 TIM-barrel motif and catalytic groove. In phenotyping and AS-PCR assays, we found that the two alleles of SNP 21881933 are significantly correlated to anthracnose resistance. Mango genotypes with the β-1,3-GLU2 allele ‘G’ showed significantly higher disease severity post-inoculation while those with β-1,3-GLU2 allele ‘A’ showed phenotypic resistance to the disease. These results indicate that β-1,3-GLU2 is involved in the primary defense response of mango against C. gloeosporioides and that SNP 21881933 enhances the activities of β-1,3-glucanases. To our knowledge, this is the first report describing the molecular basis of anthracnose resistance in mango involving β-1,3-glucanases. Furthermore, we developed a marker that can discriminate β-1,3-GLU2 SNP alleles using a simple and cost-effective allele-specific PCR assay. Our findings support the utilization of this marker for marker-assisted selection (MAS) and genotyping of resistant mangoes as early as the seedling stage. This will help improve mango breeding efficiency and significantly reduce the expenses in field inputs, maintenance, and evaluation of material over the years.

Validation of somatic cell score-associated SNPs from Holstein cattle in Sudanese Butana and Butana\u2009\xd7\u2009Holstein crossbred cattle

The Bos indicus zebu cattle Butana is the most commonly used indigenous dairy cattle breed in Sudan. In the last years, high-yielding Holstein dairy cattle were introgressed into Butana cattle to improve their milk yield and simultaneously keep their good adaption to extreme environmental conditions. With the focus on the improvement of milk production, other problems arose such as an increased susceptibility to mastitis. Thus, genetic selection for mastitis resistance should be considered to maintain healthy and productive cows. In this study, we tested 10 single nucleotide polymorphisms (SNPs) which had been associated with somatic cell score (SCS) in Holstein cattle for association with SCS in 37 purebred Butana and 203 Butana × Holstein crossbred cattle from Sudan. Animals were genotyped by competitive allele-specific PCR assays and association analysis was performed using a linear mixed model. All 10 SNPs were segregating in the crossbred Butana × Holstein populations, but only 8 SNPs in Sudanese purebred Butana cattle. The SNP on chromosome 13 was suggestively associated with SCS in the Butana × Holstein crossbred population (rs109441194, 13:79,365,467, PBF = 0.054) and the SNP on chromosome 19 was significantly associated with SCS in both populations (rs41257403, 19:50,027,458, Butana: PBF = 0.003, Butana × Holstein: PBF = 6.2 × 10−16). The minor allele of both SNPs showed an increase in SCS. Therefore, selection against the disadvantageous minor allele could be used for genetic improvement of mastitis resistance in the studied populations. However, investigations in a bigger population and across the whole genome are needed to identify additional genomic loci.

Somatic loss of the remaining allele occurs approximately in half of CHEK2-driven breast cancers and is accompanied by a border-line increase of chromosomal instability.

Germline mutations in CHEK2 gene represent the second most frequent cause of hereditary breast cancer (BC) after BRCA1/2 lesions. This study aimed to identify the molecular characteristics of CHEK2-driven BCs. Loss of heterozygosity (LOH) for the remaining CHEK2 allele was examined in 50 CHEK2-driven BCs using allele-specific PCR assays for the germline mutations and analysis of surrounding single-nucleotide polymorphisms (SNPs). Paired tumor and normal DNA samples from 25 cases were subjected to next-generation sequencing analysis. CHEK2 LOH was detected in 28/50 (56%) BCs. LOH involved the wild-type allele in 24 BCs, mutant CHEK2 copy was deleted in 3 carcinomas, while in one case the origin of the deleted allele could not be identified. Somatic PIK3CA and TP53 mutations were present in 13/25 (52%) and 4/25 (16%) tumors, respectively. Genomic features of homologous recombination deficiency (HRD), including the HRD score ≥ 42, the predominance of BRCA-related mutational signature 3, and the high proportion of long (≥ 5bp) indels, were observed only in 1/20 (5%) BC analyzed for chromosomal instability. Tumors with the deleted wild-type CHEK2 allele differed from LOH-negative cases by elevated HRD scores (median 23 vs. 7, p = 0.010) and higher numbers of chromosomal segments affected by copy number aberrations (p = 0.008). Somatic loss of the wild-type CHEK2 allele is observed in approximately half of CHEK2-driven BCs. Tumors without CHEK2 LOH are chromosomally stable. BCs with LOH demonstrate some signs of chromosomal instability; however, its degree is significantly lower as compared to BRCA1/2-associated cancers.

Development and Validation of Kompetitive Allele-Specific PCR Assays for Erucic Acid Content in Indian Mustard [Brassica juncea (L.) Czern and Coss.

Brassica juncea L. is the most widely cultivated oilseed crop in Indian subcontinent. Its seeds contain oil with very high concentration of erucic acid (≈50%). Of late, there is increasing emphasis on the development of low erucic acid varieties because of reported association of the consumption of high erucic acid oil with cardiac lipidosis. Erucic acid is synthesized from oleic acid by an elongation process involving two cycles of four sequential steps. Of which, the first step is catalyzed by β-ketoacyl-CoA synthase (KCS) encoded by the fatty acid elongase 1 (FAE1) gene in Brassica. Mutations in the coding region of the FAE1 lead to the loss of KCS activity and consequently a drastic reduction of erucic acid in the seeds. Molecular markers have been developed on the basis of variation available in the coding or promoter region(s) of the FAE1. However, majority of these markers are not breeder friendly and are rarely used in the breeding programs. Present studies were planned to develop robust kompetitive allele-specific PCR (KASPar) assays with high throughput and economics of scale. We first cloned and sequenced FAE1.1 and FAE1.2 from high and low erucic acid (<2%) genotypes of B. juncea (AABB) and its progenitor species, B. rapa (AA) and B. nigra (BB). Sequence comparisons of FAE1.1 and FAE1.2 genes for low and high erucic acid genotypes revealed single nucleotide polymorphisms (SNPs) at 8 and 3 positions. Of these, three SNPs for FAE1.1 and one SNPs for FAE1.2 produced missense mutations, leading to amino acid modifications and inactivation of KCS enzyme. We used SNPs at positions 735 and 1,476 for genes FAE1.1 and FAE1.2, respectively, to develop KASPar assays. These markers were validated on a collection of diverse genotypes and a segregating backcross progeny. KASPar assays developed in this study will be useful for marker-assisted breeding, as these can track recessive alleles in their heterozygous state with high reproducibility.

Genomic diversities of ctxB, tcpA and rstR alleles of Vibrio cholerae O139 strains isolated from Odisha, India.

The genome of Vibrio cholerae O139 strains has undergone cryptic changes since its first emergence in 1992 in South India. This study aimed to determine the presence of genotypic changes marked in ctxB, tcpA and rstR genes located within the CTX prophages among the strains of V. cholerae O139 isolated from 1999 to 2017 in Odisha. Antibiotic susceptibility test was conducted on 59 V. cholerae O139 strains. A conventional PCR assay was done for ctxB gene typing followed by sequencing along with identification of rstR and tcpA gene. Pulsed-field gel electrophoresis (PFGE) was carried out to reveal clonal variations among the V. cholerae O139 strains. Among V. cholerae O139 isolates more than 60% showed resistance to ampicillin, co-trimoxazole, furazolidone, streptomycin, neomycin and nalidixic acid. The ctxB sequencing and rstR allele-specific PCR assay revealed the presence of three genotypes 1, 3 and 4 with at least one copy of CTX Calc φ in addition to CTX ET and CTX Cl prophages in V. cholerae O139 isolates. PFGE analysis revealed 13 pulsotypes with two clades having 60% similarity among V. cholerae O139 strains. The circulating V. cholerae O139 strains in Odisha showed variation in genotypes with multiple clonal expansions over the years.

FP12.05 The Intrinsic Limitation and Clinical Impact of Mutant Allele-Specific Real-Time PCR-Based EGFR Mutation Assay in NSCLC

The detection of driver gene mutations, such as epidermal growth factor receptor (EGFR) mutations, plays a decisive role in the treatment of non-small cell lung cancer (NSCLC). Cobas EGFR mutation test is a mutant allele-specific real-time PCR assay that is commonly used in daily practice. It has a notable intrinsic detection limitation caused by its primer designs. Next-generation sequencing (NGS) is a comprehensive mutation assay that does not have the constraint. It will have a higher mutation detection rate but the clinical impact remains uncertain.

Development and validation of diagnostic SNP markers for quality control genotyping in a collection of four rice (Oryza) species

Morphological identification of closely related rice species, particularly those in the Oryza AA genome group, presents major challenges and often results in cases of misidentification. Recent work by this group identified diagnostic single nucleotide polymorphic (SNP) markers specific for several rice species and subspecies based on DArTseq next-generation sequencing technology (“DArTseq”). These SNPs can be used for quality control (QC) analysis in rice breeding and germplasm maintenance programs. Here, we present the DArTseq-based diagnostic SNPs converted into Kompetitive allele-specific PCR (KASPar or KASP) assays and validation data for a subset of them; these can be used for low-cost routine genotyping quality control (QC) analysis. Of the 224 species/subspecies’ diagnostic SNPs tested, 158 of them produced working KASP assays, a conversion success rate of 70%. Two validation experiments were run with 87 of the 158 SNP markers to ensure that the assays amplified, were polymorphic, and distinguished the five species/subspecies tested. Based on these validation test results, we recommend a panel of 36 SNP markers that clearly delineate O. barthii, O. glaberrima, O. longistaminata, O. sativa spp. indica and japonica. The KASP assays provide a flexible, rapid turnaround and cost-effective tool to facilitate germplasm curation and management of these four Oryza AA genome species across multiple genebanks.

The interaction of Epstein-Barr virus encoded transcription factor EBNA2 with multiple sclerosis risk loci is dependent on the risk genotype.

BackgroundEpstein-Barr virus (EBV) infection may be necessary for the development of Multiple sclerosis (MS). Earlier we had identified six MS risk loci that are co-located with binding sites for the EBV transcription factor Epstein-Barr Nuclear Antigen 2 (EBNA2) in EBV-infected B cells (lymphoblastoid cell lines – LCLs).MethodsWe used an allele-specific chromatin immunoprecipitation PCR assay to assess EBNA2 allelic preference. We treated LCLs with a peptide inhibitor of EBNA2 (EBNA2-TAT), reasoning that inhibiting EBNA2 function would alter gene expression at these loci if it was mediated by EBNA2.FindingsWe found that EBNA2 binding was dependent on the risk allele for five of these six MS risk loci (p < 0·05). Treatment with EBNA2-TAT significantly altered the expression of TRAF3 (p < 0·05), CD40 (p < 0·001), CLECL1 (p <0·0001), TNFAIP8 (p < 0·001) and TNFRSF1A (p < 0·001).InterpretationThese data suggest that EBNA2 can enhance or reduce expression of the gene depending on the risk allele, likely promoting EBV infection. This is consistent with the concept that these MS risk loci affect MS risk through altering the response to EBNA2. Together with the extensive data indicating a pathogenic role for EBV in MS, this study supports targeting EBV and EBNA2 to reduce their effect on MS pathogenesis.FundingFunding was provided by grants from MS Research Australia, National Health and Medical Research Council of Australia, Australian Government Research Training Program, Multiple Sclerosis International Federation, Trish Multiple Sclerosis Research Foundation.

Reversion towards benzimidazole susceptibility in Haemonchus contortus by resistance management strategies

A study was aimed to observe the impact of withdrawal of anthelmintic type and increase in refugia through community dilution on reversion towards benzimidazole (BZ) susceptibility in Haemonchus contortus at an organized sheep farm in semi-arid Rajasthan. In sheep flocks of ICAR-Central Sheep and Wool Research Institute, Avikanagar reduced efficacy (80%) on faecal egg count reduction test (FECRT) with ED50 of 0.239 μg thiabendazole (TBZ)/ml on egg hatch assay (EHA) indicative of emergence of H. contortus resistant to BZ was observed in the year 1995. Following discontinuation of BZ for deworming at farm since 1996, the efficacy varied between 0-44% in the year 2000 with predominance of BZ-resistant alleles (&gt;90%) between the year 2005 to 2010. efficacy was still low (26% on FECRT, ED50 of 0.196 μg TBZ/ml on EHA) even after 15 yr post withdrawal (in 2010). Later on, the concept of community dilution and refugia were implemented for worm management. With these strategies, during 2018-20 (22 yr post withdrawal) a significant improvement (86-93%) in efficacy of BZ was noticed against H. contortus. Simultaneously, the ED50 values ranged from 0.011 to 0.119 with an average of 0.075±0.008 μg TBZ/ml on EHA with prevalence of BZ susceptible alleles up to 48% on allele specific PCR assay. The study indicates the possibility of reversion to BZ susceptibility in H. contortus population in farm area with community dilution and refugia based worm management strategies.

Detection of EGFR mutations in liquid biopsy samples using allele-specific quantitative PCR: A comparative real-world evaluation of two popular diagnostic systems

PurposeThe detection of epidermal growth factor receptor (EGFR) mutations in plasma cell-free DNA (cfDNA) is an auxiliary tool for the molecular diagnosis of non-small cell lung cancer (NSCLC), especially when an adequate tumor tissue specimen cannot be obtained. We compared the diagnostic accuracy of two commonly used in vitro diagnostic-certified allele-specific quantitative PCR assays for detecting plasma cfDNA EGFR mutations. MethodsWe analyzed EGFR mutations in plasma cfDNA from 90 NSCLC patients (stages I–IV) before treatment (n ​= ​60) and after clinical progression on EGFR tyrosine kinase inhibitors (n ​= ​30) using the cobas EGFR mutation test v2 (Roche Molecular Systems, Inc.) and therascreen EGFR Plasma RGQ PCR kit (Qiagen GmbH). ResultsThere was higher concordance between plasma cfDNA and matched tumor tissue EGFR mutations with cobas (66.67%) compared with therascreen (55.93%). The concordance rate increased to 90.00% with cobas (Cohen's kappa coefficient, κ ​= ​0.80; p ​< ​0.0001) and 73.33% with therascreen (κ ​= ​0.49; p ​= ​0.0009) in advanced NSCLC patients. In treatment-naïve patients, cobas was superior to therascreen (sensitivity: 82.35% vs. 52.94%; specificity: 100% vs. 100%). In patients with clinical progression on EGFR tyrosine kinase inhibitors, EGFR exon 20 p.T790M was detected in 30% and 23% of cfDNA samples by cobas and therascreen, respectively. ConclusionsCobas was superior to therascreen for detection of plasma EGFR mutations in advanced NSCLC. Plasma cfDNA EGFR mutation analysis is complex; therefore, the diagnostic accuracy of commercially available assays should be validated.

Development and Exploitation of KASP Assays for Genes Underpinning Drought Tolerance Among Wheat Cultivars From Pakistan.

High-throughput genotyping for functional markers offers an excellent opportunity to effectively practice marker-assisted selection (MAS) while breeding cultivars. We developed kompetitive allele-specific PCR (KASP) assays for genes conferring drought tolerance in common wheat (Triticum aestivum L.). In total, 11 KASP assays developed in this study and five already reported assays were used for their application in wheat breeding. We investigated alleles at 16 loci associated with drought tolerance among 153 Pakistani hexaploid wheat cultivars released during 1953–2016; 28 diploid wheat accessions (16 for AA and 12 for BB) and 19 tetraploid wheat (AABB) were used to study the evolutionary history of the studied genes. Superior allelic variations of the studied genes were significantly associated with higher grain yield. Favored haplotypes of TaSnRK2.3-1A, TaSnRK2.3-1B, TaSnRK2.9-5A, TaSAP-7B, and TaLTPs-1A predominated in Pakistani wheat germplasm indicating unconscious pyramiding and selection pressure on favorable haplotypes during selection breeding. TaSnRK2.8-5A, TaDreb-B1, 1-feh w3, TaPPH-7A, TaMOC-7A, and TaPARG-2A had moderate to low frequencies of favorable haplotype among Pakistani wheat germplasm pointing toward introgression of favorable haplotypes by deploying functional markers in marker-assisted breeding. The KASP assays were compared with gel-based markers for reliability and phenotypically validated among 62 Pakistani wheat cultivars. Association analyses showed that the favorable allelic variations were significantly associated with grain yield-contributing traits. The developed molecular marker toolkit of the genes can be instrumental for the wheat breeding in Pakistan.

Development and validation of SNP genotyping assays to identify genetic sex in the swimming crab Portunus trituberculatus

The swimming crab Portunus trituberculatus is an economically vital aquaculture species in China and South East Asia. Monosex female culture has been identified as a key priority due to their increased market value, consumer preference and enhanced growth performance. However, the lack of a rapid and reliable sex-linked marker panel has hampered the application of sex control and understanding of the sex determination in the species. In this study, two sex-linked SNPs obtained from our simplified genomic data were validated by sequencing and then converted into competitive allele-specific PCR assays. The results showed high consistency between the genetic and phenotypic sex providing therefore a rapid test sex identification. Moreover, heterogametic genotypes in males provided a significant genetic evidence to support an XX/XY sex determination system in the P. trituberculatus.

QTL mapping of seedling biomass and root traits under different nitrogen conditions in bread wheat (Triticum aestivum L.)

Plant nitrogen assimilation and use efficiency in the seedling's root system are beneficial for adult plants in field condition for yield enhancement. Identification of the genetic basis between root traits and N uptake plays a crucial role in wheat breeding. In the present study, 198 doubled haploid lines from the cross of Yangmai 16/Zhongmai 895 were used to identify quantitative trait loci (QTLs) underpinning four seedling biomass traits and five root system architecture (RSA) related traits. The plants were grown under hydroponic conditions with control, low and high N treatments (Ca(NO3)2·4H2O at 0, 0.05 and 2.0 mmol L−1, respectively). Significant variations among the treatments and genotypes, and positive correlations between seedling biomass and RSA traits (r=0.20 to 0.98) were observed. Inclusive composite interval mapping based on a high-density map from the Wheat 660K single nucleotide polymorphisms (SNP) array identified 51 QTLs from the three N treatments. Twelve new QTLs detected on chromosomes 1AL (1) in the control, 1DS (2) in high N treatment, 4BL (5) in low and high N treatments, and 7DS (3) and 7DL (1) in low N treatments, are first reported in influencing the root and biomass related traits for N uptake. The most stable QTLs (RRS.caas-4DS) on chromosome 4DS, which were related to ratio of root to shoot dry weight trait, was in close proximity of the Rht-D1 gene, and it showed high phenotypic effects, explaining 13.1% of the phenotypic variance. Twenty-eight QTLs were clustered in 12 genetic regions. SNP markers tightly linked to two important QTLs clusters C10 and C11 on chromosomes 6BL and 7BL were converted to kompetitive allele-specific PCR (KASP) assays that underpin important traits in root development, including root dry weight, root surface area and shoot dry weight. These QTLs, clusters and KASP assays can greatly improve the efficiency of selection for root traits in wheat breeding programmes.