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

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).

scholarly journals Linkage and Association Mapping Identifies Loci and Develops Kompetitive Allele Specific PCR Markers for Improving Wheat Grain Protein Content

2021 ◽  
Author(s):  
Peng Jiang ◽  
Peng Zhang ◽  
Lei Wu ◽  
Yi He ◽  
Chang Li ◽  
...  
Keyword(s):  
Association Mapping ◽  
Protein Content ◽  
Large Scale ◽  
Grain Protein Content ◽  
Grain Protein ◽  
Wheat Grain ◽  
Breeding Lines ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

Abstract Wheat grain protein content (GPC) is an important quality indicator. The GPC of wheat grown in the middle and lower reaches of the Yangtze River is often low. Marker-assisted selection (MAS) is an effective tool for improving quantitative traits; however, except Gpc-B1, most markers have not been effectively applied in GPC improvement, although many related loci have been identified. Linkage analysis using a recombinant inbred line population from the cross of core parents of Ningmai 9 and Yangmai 158 and association mapping using the local cultivated varieties were performed and nine candidate intervals were identified. The appropriate kompetitive allele specific PCR (KASP) markers associated with GPC were successfully developed and applied in 1163 F4 breeding lines. Three markers, Kgpc-2B, Kgpc-2D, and Kgpc-4A, were validated to be significantly related to GPC by large-scale association mapping, and they were combined to achieve the highest efficiency to enhance GPC. We applied these markers in 164 F6 breeding lines and obtained 15 lines with high GPC, indicating their high selective efficiency. Further, strategies for gene exploration in the three significant intervals were proposed. These results were expected to provide a novel route for improving GPC in wheat quality breeding.

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

Agronomy ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 2315
Author(s):  
Moctar Kante ◽  
Hannele Lindqvist-Kreuze ◽  
Leticia Portal ◽  
Maria David ◽  
Manuel Gastelo
Keyword(s):  
Quality Control ◽  
Low Cost ◽  
Yield Reduction ◽  
Genetic Gains ◽  
Specific Pcr ◽  
Breeding Populations ◽  
Allele Specific ◽  
Cultivated Potatoes ◽  
Allele Specific Pcr ◽  
Kasp Markers

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.

scholarly journals Developing KASP Markers for Identification of Basmati Rice Varieties

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.

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

Plants ◽  
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.

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

Agronomy ◽  
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.

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

2020 ◽  
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.

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

1996 ◽  
Vol 75 (05) ◽  
pp. 757-759 ◽  
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.

scholarly journals Development of genic KASP SNP markers from RNA-Seq data for map-based cloning and marker-assisted selection in maize

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zhengjie Chen ◽  
Dengguo Tang ◽  
Jixing Ni ◽  
Peng Li ◽  
Le Wang ◽  
...  
Keyword(s):  
Marker Assisted Selection ◽  
Inbred Lines ◽  
Average Density ◽  
Snp Markers ◽  
Data Sets ◽  
Rna Seq ◽  
Specific Pcr ◽  
Maize Inbred Lines ◽  
Allele Specific ◽  
Allele Specific Pcr

Abstract Background Maize is one of the most important field crops in the world. Most of the key agronomic traits, including yield traits and plant architecture traits, are quantitative. Fine mapping of genes/ quantitative trait loci (QTL) influencing a key trait is essential for marker-assisted selection (MAS) in maize breeding. However, the SNP markers with high density and high polymorphism are lacking, especially kompetitive allele specific PCR (KASP) SNP markers that can be used for automatic genotyping. To date, a large volume of sequencing data has been produced by the next generation sequencing technology, which provides a good pool of SNP loci for development of SNP markers. In this study, we carried out a multi-step screening method to identify kompetitive allele specific PCR (KASP) SNP markers based on the RNA-Seq data sets of 368 maize inbred lines. Results A total of 2,948,985 SNPs were identified in the high-throughput RNA-Seq data sets with the average density of 1.4 SNP/kb. Of these, 71,311 KASP SNP markers (the average density of 34 KASP SNP/Mb) were developed based on the strict criteria: unique genomic region, bi-allelic, polymorphism information content (PIC) value ≥0.4, and conserved primer sequences, and were mapped on 16,161 genes. These 16,161 genes were annotated to 52 gene ontology (GO) terms, including most of primary and secondary metabolic pathways. Subsequently, the 50 KASP SNP markers with the PIC values ranging from 0.14 to 0.5 in 368 RNA-Seq data sets and with polymorphism between the maize inbred lines 1212 and B73 in in silico analysis were selected to experimentally validate the accuracy and polymorphism of SNPs, resulted in 46 SNPs (92.00%) showed polymorphism between the maize inbred lines 1212 and B73. Moreover, these 46 polymorphic SNPs were utilized to genotype the other 20 maize inbred lines, with all 46 SNPs showing polymorphism in the 20 maize inbred lines, and the PIC value of each SNP was 0.11 to 0.50 with an average of 0.35. The results suggested that the KASP SNP markers developed in this study were accurate and polymorphic. Conclusions These high-density polymorphic KASP SNP markers will be a valuable resource for map-based cloning of QTL/genes and marker-assisted selection in maize. Furthermore, the method used to develop SNP markers in maize can also be applied in other species.

scholarly journals Two User-Friendly Molecular Markers Developed for the Identification of Hybrid Lethality Genes in Brassica oleracea

Agronomy ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 982
Author(s):  
Zhiliang Xiao ◽  
Congcong Kong ◽  
Fengqing Han ◽  
Limei Yang ◽  
Mu Zhuang ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Brassica Oleracea ◽  
Rapid Identification ◽  
Inbred Lines ◽  
Hybrid Lethality ◽  
Specific Pcr ◽  
Allele Specific ◽  
User Friendly ◽  
Allele Specific Pcr ◽  
Kasp Marker

Cabbage (Brassica oleracea) is an important vegetable crop that is cultivated worldwide. Previously, we reported the identification of two dominant complementary hybrid lethality (HL) genes in cabbage that could result in the death of hybrids. To avoid such losses in the breeding process, we attempted to develop molecular markers to identify HL lines. Among 54 previous mapping markers closely linked to BoHL1 or BoHL2, only six markers for BoHL2 were available in eight cabbage lines (two BoHL1 lines; three BoHL2 lines; three lines without BoHL); however, they were neither universal nor user-friendly in more inbred lines. To develop more accurate markers, these cabbage lines were resequenced at an ~20× depth to obtain more nucleotide variations in the mapping regions. Then, an InDel in BoHL1 and a single-nucleotide polymorphism (SNP) in BoHL2 were identified, and the corresponding InDel marker MBoHL1 and the competitive allele-specific PCR (KASP) marker KBoHL2 were developed and showed 100% accuracy in eight inbred lines. Moreover, we identified 138 cabbage lines using the two markers, among which one inbred line carried BoHL1 and 11 inbred lines carried BoHL2. All of the lethal line genotypes obtained with the two markers matched the phenotype. Two markers were highly reliable for the rapid identification of HL genes in cabbage.

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