4,207 research outputs found
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Use of genetic markers for the detection of off-types for DUS phenotypic traits in the inbreeding crop, barley
Detection of crop off-types is of interest for
multiple uses, including the assessment of uniformity
for new plant variety applications during distinctness,
uniformity and stability (DUS) testing for the awarding
of plant breeders’ rights (PBR). Here, we investigate
whether genetic markers, in this case Kompetitive
Allele-Specific PCR (KASP), can be used for the identification
off-types for phenotypes assessed for DUS in
the inbreeding cereal crop, barley (Hordeum vulgare).
To demonstrate proof of principle, KASP markers diagnostic
for phenotypic expression of nine DUS phenotypes,
and DNA from two barley varieties (‘Pelican’ and
‘Felicie’) carrying contrasting alleles at each marker
were used. We found that for the majority of markers,
it was possible to robustly call alleles down to template
DNA concentrations of 2 ng, but not ≤ 0.2 ng. When
used in mixtures of DNA consisting of ‘Felicie’ DNA
spiked with different concentrations of ‘Pelican’ DNA,
robust allele calling was possible in DNA mixtures
down to 18 ng:2 ng. Collectively, this demonstrates that
where diagnostic markers are available, molecular identification
of a single off-type for a given DUS trait
within a bulk of ten individuals should be possible. We
validated this assumption, with all of the diagnostic
genetic markers investigated found to robustly detect
DUS off-types at a frequency of 10% in DNA extracted
from tissue collected from pools of 10 individuals. Ultimately,
this work demonstrates that, where diagnostic
polymorphisms are known for DUS traits, KASP
markers should be able to robustly detect off-types or
cross-contamination within DNA samples from a diploid
inbred species down to 10%. While just two varieties
that contrasted for the eight DUS targeted were
investigated in this study, as the markers used are diagnostic
for their relevant phenotype (or a proportion of
the variation observed for that phenotype), in theory the
approach should be valid for any variety studied—
although the introduction of novel alleles via spontaneous
mutation or more exotic germplasm pools may
mean that marker sets would need to be periodically
added to or updated. However, we nevertheless demonstrate
the principle that, for a subset of DUS traits,
molecular markers can now be robustly used as a tool
towards determining all three components of the DUS
testing process in barley. These results are relevant for
the assessment of varietal uniformity by crop breeders,
crop testing authorities and germplasm maintenance, as
well as highlighting the potential use of bulk samples
rather than individual plant samples for assessment of
distinctness by molecular methods
Methods for Scarless, Selection-Free Generation of Human Cells and Allele-Specific Functional Analysis of Disease-Associated SNPs and Variants of Uncertain Significance.
With the continued emergence of risk loci from Genome-Wide Association studies and variants of uncertain significance identified from patient sequencing, better methods are required to translate these human genetic findings into improvements in public health. Here we combine CRISPR/Cas9 gene editing with an innovative high-throughput genotyping pipeline utilizing KASP (Kompetitive Allele-Specific PCR) genotyping technology to create scarless isogenic cell models of cancer variants in ~1 month. We successfully modeled two novel variants previously identified by our lab in the PALB2 gene in HEK239 cells, resulting in isogenic cells representing all three genotypes for both variants. We also modeled a known functional risk SNP of colorectal cancer, rs6983267, in HCT-116 cells. Cells with extremely low levels of gene editing could still be identified and isolated using this approach. We also introduce a novel molecular assay, ChIPnQASO (Chromatin Immunoprecipitation and Quantitative Allele-Specific Occupation), which uses the same technology to reveal allele-specific function of these variants at the DNA-protein interaction level. We demonstrated preferential binding of the transcription factor TCF7L2 to the rs6983267 risk allele over the non-risk. Our pipeline provides a platform for functional variant discovery and validation that is accessible and broadly applicable for the progression of efforts towards precision medicine
Exploring the Importance of Single Nucleotide Polymorphisms of HSPA9 in DNA of Sarcoma Patients
The aim of this project was to identify genetic variants that may influence the risk and progression of sarcoma through targeted genotyping of HSPA9 gene. It is important to look at genetic variants in DNA samples because if a variant is determined to be more likely than another, a screening for the particular variant can be done to identify a patient’s risk of sarcoma. The study population was sarcoma patients from the International Sarcoma Kindred Study. These patients had no mutations in p53 or MDM2. Genotyping data from the HapMap project (hapmap.org) for HSPA9 was used to identify the polymorphisms needed to tag the entire region. In order to genotype the DNA sample, KASP reagents (KBioSciences, UK) were used. KASP uses a two-set PCR process. Allele specific primers are used to preferentially amplify each allele of a given SNP. The specific genetic variations of HSPA9 in sarcoma patient DNA samples with no mutations in p53 or MDM2 amplification are not more or less likely to occur than in DNA samples with the mutation or amplification. If continued research can show that MDM2 is not amplified, but activated through other mechanisms such as the interaction between polymorphisms of mitochondrial genes, p53, or MDM2, we can propose anti-MDM2 therapies to the patients with these polymorphisms
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Three previously characterized resistances to yellow rust are encoded by a single locus Wtk1.
The wild emmer wheat (Triticum turgidum ssp. dicoccoides; WEW) yellow (stripe) rust resistance genes Yr15, YrG303, and YrH52 were discovered in natural populations from different geographic locations. They all localize to chromosome 1B but were thought to be non-allelic based on differences in resistance response. We recently cloned Yr15 as a Wheat Tandem Kinase 1 (WTK1) and show here that these three resistance loci co-segregate in fine-mapping populations and share an identical full-length genomic sequence of functional Wtk1. Independent ethyl methanesulfonate (EMS)-mutagenized susceptible yrG303 and yrH52 lines carried single nucleotide mutations in Wtk1 that disrupted function. A comparison of the mutations for yr15, yrG303, and yrH52 mutants showed that while key conserved residues were intact, other conserved regions in critical kinase subdomains were frequently affected. Thus, we concluded that Yr15-, YrG303-, and YrH52-mediated resistances to yellow rust are encoded by a single locus, Wtk1. Introgression of Wtk1 into multiple genetic backgrounds resulted in variable phenotypic responses, confirming that Wtk1-mediated resistance is part of a complex immune response network. WEW natural populations subjected to natural selection and adaptation have potential to serve as a good source for evolutionary studies of different traits and multifaceted gene networks
Genetic mapping of legume orthologs reveals high conservation of synteny between lentil species and the sequenced genomes of Medicago and chickpea.
Lentil (Lens culinaris Medik.) is a global food crop with increasing importance for food security in south Asia and other regions. Lens ervoides, a wild relative of cultivated lentil, is an important source of agronomic trait variation. Lens is a member of the galegoid clade of the Papilionoideae family, which includes other important dietary legumes such as chickpea (Cicer arietinum) and pea (Pisum sativum), and the sequenced model legume Medicago truncatula. Understanding the genetic structure of Lens spp. in relation to more fully sequenced legumes would allow leveraging of genomic resources. A set of 1107 TOG-based amplicons were identified in L. ervoides and a subset thereof used to design SNP markers for mapping. A map of L. ervoides consisting of 377 SNP markers spread across seven linkage groups was developed using a GoldenGate genotyping array and single SNP marker assays. Comparison with maps of M. truncatula and L. culinaris documented considerable shared synteny and led to the identification of a few major translocations and a major inversion that distinguish Lens from M. truncatula, as well as a translocation that distinguishes L. culinaris from L. ervoides. The identification of chromosome-level differences among Lens spp. will aid in the understanding of introgression of genes from L. ervoides into cultivated L. culinaris, furthering genetic research and breeding applications in lentil
Large Scale Spectral Clustering Using Approximate Commute Time Embedding
Spectral clustering is a novel clustering method which can detect complex
shapes of data clusters. However, it requires the eigen decomposition of the
graph Laplacian matrix, which is proportion to and thus is not
suitable for large scale systems. Recently, many methods have been proposed to
accelerate the computational time of spectral clustering. These approximate
methods usually involve sampling techniques by which a lot information of the
original data may be lost. In this work, we propose a fast and accurate
spectral clustering approach using an approximate commute time embedding, which
is similar to the spectral embedding. The method does not require using any
sampling technique and computing any eigenvector at all. Instead it uses random
projection and a linear time solver to find the approximate embedding. The
experiments in several synthetic and real datasets show that the proposed
approach has better clustering quality and is faster than the state-of-the-art
approximate spectral clustering methods
Isolation and fine mapping of Rps6: An intermediate host resistance gene in barley to wheat stripe rust
A plant may be considered a nonhost of a pathogen if all known genotypes of a plant species are resistant to all known isolates of a pathogen species. However, if a small number of genotypes are susceptible to some known isolates of a pathogen species this plant maybe considered an intermediate host. Barley (Hordeum vulgare) is an intermediate host for Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust. We wanted to understand the genetic architecture underlying resistance to Pst and to determine whether any overlap exists with resistance to the host pathogen, Puccinia striiformis f. sp. hordei (Psh). We mapped Pst resistance to chromosome 7H and show that host and intermediate host resistance is genetically uncoupled. Therefore, we designate this resistance locus Rps6. We used phenotypic and genotypic selection on F2:3 families to isolate Rps6 and fine mapped the locus to a 0.1 cM region. Anchoring of the Rps6 locus to the barley physical map placed the region on two adjacent fingerprinted contigs. Efforts are now underway to sequence the minimal tiling path and to delimit the physical region harbouring Rps6. This will facilitate additional marker development and permit identification of candidate genes in the region
Genetic diversity and linkage disequilibrium using SNP (KASP) and AFLP markers in a worldwide durum wheat (Triticum turgidum L. Var durum) collection
The aim of this work was to analyze the genetic diversity and linkage disequilibrium in a collection of 168 durum wheat accessions (Triticum turgidum L. var. durum) of different origins. Our collection was mainly composed of released and unreleased Argentinian germplasm, with additional genotypes from Italy, Chile, France, CIMMYT, Cyprus, USA and WANA region. To this end, the entire collection was characterized with 85 Single Nucleotide Polymorphism (SNP) markers obtained by Kompetitive Allele Specific PCR (KASP), giving a heterozygosity (He) mean value of 0.183 and a coefficient of genetic differentiation (Gst) value of 0.139. A subset of 119 accessions was characterized with six Amplified Fragment Length Polymorphism (AFLP) primer combinations. A total of 181 polymorphic markers (125 AFLP and 56 SNP) amplified across this subset revealed He measures of 0.352 and 0.182, respectively. Of these, 134 were selected to estimate the genome-wide linkage disequilibrium obtaining low significant values (r2 = 0.11) in the subset, indicating its suitability for future genome-wide association studies (GWAS). The structure analysis conducted in the entire collection with SNP detected two subpopulations. However, the structure analysis conducted with AFLP markers in the subset of 119 accessions proved to have greater degree of resolution and detect six subpopulations. The information provided by both marker types was complementary and showed a strong association between old Argentinian and Italian germplasm and a contribution of CIMMYT germplasm to modern Argentinian, Chilean and Cypriot accessions. The influence of Mediterranean germplasm, mainly from Italy, on part of the modern Argentinian cultivars or breeding lines was also clearly evidenced. Although our analysis yields conclusive results and useful information for association mapping studies, further analyses are needed to refine the number of subpopulations present in the germplasm collection analyzed.Fil: Roncallo, Pablo Federico. Consejo Nacional de Investigaciones CientÃficas y Técnicas. Centro CientÃfico Tecnológico Conicet - BahÃa Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; ArgentinaFil: Beaufort, Valeria. Consejo Nacional de Investigaciones CientÃficas y Técnicas. Centro CientÃfico Tecnológico Conicet - BahÃa Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; ArgentinaFil: Larsen, Adelina Olga. Instituto Nacional de TecnologÃa Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Barrow; ArgentinaFil: Dreisigacker, Susanne. Centro Internacional de Mejoramiento de Maiz y Trigo; MéxicoFil: Echenique, Carmen Viviana. Consejo Nacional de Investigaciones CientÃficas y Técnicas. Centro CientÃfico Tecnológico Conicet - BahÃa Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentin
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