Kompetitive Allele Specific PCR (KASP) Markers for Potato: An Effective Tool for Increased Genetic Gains

Potato virus Y (PVY) and Phytophthora infestans (Mont.) de Bary that causes potato late blight (LB), pose serious constraints to cultivated potatoes due to significant yield reduction, and phenotyping for resistance remains challenging. Breeding operations for vegetatively propagated crops can lead to genotype mislabeling that, in turn, reduces genetic gains. Low-density and low-cost molecular marker assessment for phenotype prediction and quality control is a viable option for breeding programs. Here, we report on the development of kompetitive allele specific PCR (KASP) markers for LB and PVY resistance, and for routine quality control assessment of different breeding populations. Two KASP markers for LB resistance and two for PVY Ryadg were validated with an estimated assay power that ranged between 0.65 and 0.88. The developed QC KASP markers demonstrated the capability of discriminating tetraploid calls in breeding materials, including full-sibs and half-sibs. Routine implementation of the developed markers in a breeding program would assist with better allocation of resources and enable precise characterization of breeding material, thereby leading to increased genetic gains.

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Towards low cost, multiplex clinical genotyping: 4-fluorescent Kompetitive Allele-Specific PCR and its application on pharmacogenetics

PLoS ONE ◽

10.1371/journal.pone.0230445 ◽

2020 ◽

Vol 15 (3) ◽

pp. e0230445 ◽

Cited By ~ 1

Author(s):

Wei Suo ◽

Xiujin Shi ◽

Sha Xu ◽

Xiao Li ◽

Yang Lin

Keyword(s):

Low Cost ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

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Single tube allele specific PCR: a low cost technique for molecular screening of sickle cell anaemia in Nigeria

African Health Sciences ◽

10.4314/ahs.v18i4.20 ◽

2018 ◽

Vol 18 (4) ◽

pp. 995

Author(s):

Emuejevoke T Toye ◽

Guido Van Marle ◽

Wendy Hutchins ◽

Olayinka Abgabiaje ◽

Joy Okpuzor Okpuzor

Keyword(s):

Sickle Cell ◽

Sickle Cell Anaemia ◽

Low Cost ◽

Molecular Screening ◽

Single Tube ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

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Comparison of Kompetitive Allele Specific PCR (KASP) and genotyping by sequencing (GBS) for quality control analysis in maize

BMC Genomics ◽

10.1186/s12864-015-2180-2 ◽

2015 ◽

Vol 16 (1) ◽

Cited By ~ 35

Author(s):

Berhanu Tadesse Ertiro ◽

Veronica Ogugo ◽

Mosisa Worku ◽

Biswanath Das ◽

Michael Olsen ◽

...

Keyword(s):

Quality Control ◽

Genotyping By Sequencing ◽

Control Analysis ◽

Specific Pcr ◽

Quality Control Analysis ◽

Allele Specific ◽

Allele Specific Pcr

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Limits of Dideoxysequencing in the Detection of Somatic Mutations in Gastrointestinal Stromal Tumors

Acta Medica Martiniana ◽

10.1515/acm-2015-0013 ◽

2015 ◽

Vol 15 (3) ◽

pp. 13-20 ◽

Cited By ~ 1

Author(s):

K Jasek ◽

V Buzalkova ◽

P Szepe ◽

L Plank ◽

Z Lasabova

Keyword(s):

Gastrointestinal Stromal Tumors ◽

Somatic Mutations ◽

Low Cost ◽

Gene Mutations ◽

Wild Type ◽

Stromal Tumors ◽

Specific Pcr ◽

Allele Specific ◽

Detection Of Mutations ◽

Allele Specific Pcr

Abstract Detection of mutations in cancer is particularly important in terms of proper treatment and targeted therapy. The aim of this study was the comparison of two methods: allele-specific PCR (AS-PCR) and dideoxysequencing applied for the identification of BRAF gene mutations in wild-type gastrointestinal stromal tumors (WT GISTs). We have optimized the conditions for the detection V600E mutation representing the c.1799 T>A substitution by AS-PCR and have used dideoxysequencing to verify our results. In nine cases, we were able to detect the mutation by AS-PCR approach; however, the mutations have been confirmed by dideoxysequencing in four cases only. AS-PCR is fast and low cost method for the detection of V600E mutation which was validated as a sensitive assay for the identification of the most common BRAF mutation in DNA extracted from paraffin-embedded tissue of WT GISTs.

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Developing KASP Markers for Identification of Basmati Rice Varieties

Food Analytical Methods ◽

10.1007/s12161-020-01892-3 ◽

2020 ◽

Author(s):

Katherine Steele ◽

Mark Quinton Tulloch ◽

Malcolm Burns ◽

Werner Nader

Keyword(s):

Microsatellite Markers ◽

Rice Genome ◽

Basmati Rice ◽

Nucleotide Polymorphisms ◽

Rice Varieties ◽

Specific Pcr ◽

Wide Range ◽

Allele Specific ◽

Allele Specific Pcr ◽

Kasp Markers

Abstract Authentication of Basmati rice has relied on microsatellite markers since 2004, but microsatellites cannot distinguish between all of the forty-one Basmati varieties approved in 2017. This study investigated whether single nucleotide polymorphisms (SNP) and insertion/deletion (InDel) variations developed into KASP™ (Kompetitive Allele Specific PCR; LGC Biosearch Technologies) could be used to distinguish between commercial Basmati varieties. Suitable loci were identified by comparing whole genome sequences of 120 diverse rice accessions. Sequences flanking these loci were standardized across a wide range of rice genomes to produce optimal KASP designs. We selected 364 KASP designs to use for genotyping; they were either near to informative microsatellite markers, within the Badh2 and Waxy genes, or distributed throughout the rice genome. Genotypes for 327 KASP were obtained with 255 loci revealing polymorphism in up to 41 samples of approved Basmati varieties and 20 non-Basmati varieties. The varieties genotyped had not been used in the KASP design process. KASP were able to distinguish between commercial Basmati varieties that could not be distinguished with currently available microsatellites. Thirty-seven Basmati varieties could be distinguished from all others with between 3 and 8 KASP markers out of a pool of 98 informative markers. A reduced set of 24 KASP markers could determine whether a sample belongs to one of eight family groups. All of the KASP markers used in this study can be purchased from LGC Biosearch Technologies. These markers have potential to be used by industry for routine testing and regulation.

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Identification of Accession-Specific Variants and Development of KASP Markers for Assessing the Genetic Makeup of Crop Seeds

10.21203/rs.3.rs-1119082/v1 ◽

2021 ◽

Author(s):

Seongmin Hong ◽

Su Ryun Choi ◽

Jihyeong Kim ◽

Young-Min Jeong ◽

Suk-Yoon Kwon ◽

...

Keyword(s):

Core Collection ◽

F1 Hybrids ◽

Genetic Purity ◽

Breeding Lines ◽

Seed Testing ◽

Specific Pcr ◽

Allele Specific ◽

Genetic Makeup ◽

Allele Specific Pcr ◽

Kasp Markers

Abstract Background Most crop seeds are F1 hybrids. Seed providers and plant breeders must be confident that the seed supplied to growers is of known, and uniform, genetic makeup. This requires maintenance of pure genotypes of the parental lines and testing to ensure the genetic purity of the F1 seed. Traditionally, seed testing for purity was done with a grow-out test (GOT) in the field, but these tests are time consuming and costly. Seed testing with molecular markers was introduced as a replacement for GOT early in the last decade. Recently, Kompetitive allele specific PCR (KASP) markers are promising tools for genetic testing of seeds. However, the markers available at that time could be inaccurate and could be used with only a small number of accessions or varieties due to the limited genetic information and reference genomes available. Results Here, we identified 4,925,742 SNPs in 50 accessions of the Brasscia rapa core collection. Furthermore, the total 2,925 SNPs were selected as accession-specific SNPs, considering properties of flanking region harboring accession-specific SNPs and genic region conservation among accessions by NGS analysis. In total, 100 accession-specific markers were developed as accession-specific KASP markers. Based on the results of our validation experiments, the accession-specific markers successfully distinguish individuals from the mixed population including 50 target accessions from B. rapa core collection and outgroup. Conclusions This study provides efficient methods for developing KASP markers to distinguish individuals from the mixture comprised of breeding lines and germplasms from the resequencing data of Chinese cabbage (Brassica rapa spp. pekinensis).

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Development of 454 New Kompetitive Allele-Specific PCR (KASP) Markers for Temperate japonica Rice Varieties

Plants ◽

10.3390/plants9111531 ◽

2020 ◽

Vol 9 (11) ◽

pp. 1531

Author(s):

Kyeong-Seong Cheon ◽

Young-Min Jeong ◽

Hyoja Oh ◽

Jun Oh ◽

Do-Yu Kang ◽

...

Keyword(s):

Agronomic Traits ◽

High Impact ◽

Japonica Rice ◽

Rice Varieties ◽

Breeding Programs ◽

Temperate Japonica ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr ◽

Kasp Markers

Temperate japonica rice varieties exhibit wide variation in the phenotypes of several important agronomic traits, including disease resistance, pre-harvest sprouting resistance, plant architecture, and grain quality, indicating the presence of genes contributing to favorable agronomic traits. However, gene mapping and molecular breeding has been hampered as a result of the low genetic diversity among cultivars and scarcity of polymorphic DNA markers. Single nucleotide polymorphism (SNP)-based kompetitive allele-specific PCR (KASP) markers allow high-throughput genotyping for marker-assisted selection and quantitative trait loci (QTL) mapping within closely related populations. Previously, we identified 740,566 SNPs and developed 771 KASP markers for Korean temperate japonica rice varieties. However, additional markers were needed to provide sufficient genome coverage to support breeding programs. In this study, the 740,566 SNPs were categorized according to their predicted impacts on gene function. The high-impact, moderate-impact, modifier, and low-impact groups contained 703 (0.1%), 20,179 (2.7%), 699,866 (94.5%), and 19,818 (2.7%) SNPs, respectively. A subset of 357 SNPs from the high-impact group was selected for initial KASP marker development, resulting in 283 polymorphic KASP markers. After incorporation of the 283 markers with the 771 existing markers in a physical map, additional markers were developed to fill genomic regions with large gaps between markers, and 171 polymorphic KASP markers were successfully developed from 284 SNPs. Overall, a set of 1225 KASP markers was produced. The markers were evenly distributed across the rice genome, with average marker density of 3.3 KASP markers per Mbp. The 1225 KASP markers will facilitate QTL/gene mapping and marker-assisted selection in temperate japonica rice breeding programs.

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Complete Mitochondrial Genome and a Set of 10 Novel Kompetitive Allele-Specific PCR Markers in Ginseng (Panax ginseng C. A. Mey.)

Agronomy ◽

10.3390/agronomy10121868 ◽

2020 ◽

Vol 10 (12) ◽

pp. 1868

Author(s):

Woojong Jang ◽

Hyun Oh Lee ◽

Jang-Uk Kim ◽

Jung-Woo Lee ◽

Chi-Eun Hong ◽

...

Keyword(s):

Mitochondrial Genome ◽

Panax Ginseng ◽

Complete Mitochondrial Genome ◽

Perennial Herb ◽

Specific Pcr ◽

Mutational Hotspots ◽

Allele Specific ◽

Complete Mitogenome ◽

Allele Specific Pcr ◽

Kasp Markers

Panax ginseng C. A. Mey., a perennial herb belonging to the family Araliaceae, is a valuable medicinal plant with distinctive biological characteristics. However, comprehensive analyses of the mitochondrial genome (mitogenome) are lacking. In this study, we sequenced the complete mitogenome of ginseng based on long-read data from the Nanopore sequencing platform. The mitogenome was assembled into a “master circle” form of 464,705 bp and contained 72 unique genes. The genome had three large repeat regions, and 10.42% of the sequences were mitogenome sequences of plastid origin (MTPTs). In total, 278 variants (213 SNPs and 65 InDels) were discovered, most of which were identified in intergenic regions. The MTPT regions were mutational hotspots, harboring 74.5% of the variants. The ginseng mitogenome showed a higher mutation rate than that of the chloroplast genome, and this pattern is uncommon in plants. In addition, 10 Kompetitive allele-specific PCR (KASP) markers were developed from 10 SNPs, excluding those in MTPT regions. These markers accurately identified the genotypes of 59 Korean ginseng accessions and elucidated mitogenome diversity. These results provide insight into organellar genomes and genetic diversity in ginseng. Moreover, the complete mitogenome sequence and 10 KASP markers will be useful for ginseng research and breeding.

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Development of Wheat-Aegilops caudata Introgression Lines and Their Characterisation Using Genome-Specific KASP Markers

10.1101/2020.03.03.975326 ◽

2020 ◽

Cited By ~ 1

Author(s):

Surbhi Grewal ◽

Manel Othmeni ◽

Jack Walker ◽

Stella Hubbart Edwards ◽

Cai-yun Yang ◽

...

Keyword(s):

Chromosomal Rearrangements ◽

Resistance Factors ◽

Specific Pcr ◽

Wheat Improvement ◽

Genome Wide ◽

Allele Specific ◽

Aegilops Caudata ◽

Allele Specific Pcr ◽

Kasp Markers

ABSTRACTAegilops caudata L. [syn. Ae. markgrafii (Greuter) Hammer], a diploid wild relative of wheat (2n = 2x = 14, CC), is an important source for new genetic variation for wheat improvement due to a variety of disease resistance factors along with tolerance for various abiotic stresses. Its practical utilisation in wheat improvement can be facilitated through the generation of genome-wide introgressions leading to a variety of different wheat–Ae. caudata recombinant lines. In this study, we report the generation of nine such wheat–Ae. caudata recombinant lines which were characterized using wheat genome-specific KASP (Kompetitive Allele Specific PCR) markers and multi-colour genomic in situ hybridization (mcGISH). Of these, six lines have stable homozygous introgressions from Ae. caudata and will be used for future trait analysis. Through a combination of molecular and cytological analysis of all the recombinant lines, we were able to physically map 182 KASP markers onto the seven Ae. caudata chromosomes, of which 155 were polymorphic specifically with only one wheat subgenome. Comparative analysis of the physical positions of these markers in the Ae. caudata and wheat genomes confirmed that the former had chromosomal rearrangements with respect to wheat, as previously reported. These wheat–Ae. caudata recombinant lines and KASP markers provide a useful genetic resource for wheat improvement with the latter having a wider impact as a tool for detection of introgressions from other Aegilops species into wheat.

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Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650362 ◽

1996 ◽

Vol 75 (05) ◽

pp. 757-759 ◽

Cited By ~ 11

Author(s):

Rainer Blasczyk ◽

Markus Ritter ◽

Christian Thiede ◽

Jenny Wehling ◽

Günter Hintz ◽

...

Keyword(s):

Direct Sequencing ◽

Activated Protein C ◽

Factor V Leiden ◽

Pcr Amplification ◽

Factor V ◽

Specific Pcr ◽

Pcr Rflp ◽

Allele Specific ◽

Specific Amplification ◽

Allele Specific Pcr

SummaryResistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

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