Citrus (Rutaceae) SNP Markers Based on Competitive Allele-Specific PCR; Transferability Across the Aurantioideae Subfamily

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.

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Validation of SNP markers associated with ascochyta blight resistance in pea

Canadian Journal of Plant Science ◽

10.1139/cjps-2018-0211 ◽

2019 ◽

Vol 99 (2) ◽

pp. 243-249

Author(s):

Ambuj B. Jha ◽

Krishna K. Gali ◽

Sabine Banniza ◽

Thomas D. Warkentin

Keyword(s):

Association Studies ◽

Ascochyta Blight ◽

Snp Markers ◽

Blight Resistance ◽

Maturity Stage ◽

Single Nucleotide ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr ◽

Important Disease

Ascochyta blight of pea is an important disease that can cause severe yield loss. Our previous studies identified several closely linked single nucleotide polymorphism (SNP) markers associated with ascochyta blight resistance. The objective of this study was to validate SNP markers in 36 cultivars from the Saskatchewan pea regional variety trial. Ascochyta blight scores ranged from 1.0 to 9.0 at the physiological maturity stage under field conditions across the 25 site–years in Saskatchewan from 2013 to 2017. Based on Kompetitive Allele-Specific PCR assays, six SNP markers were used for an association study. SNP markers RGA-G3Ap103, PsC8780p118, and PsC22609p103 were significantly (P < 0.05) associated with ascochyta blight scores in 2013 and 2016 at Saskatoon. PsC8780p118 was significantly associated with ascochyta blight scores at Milden in 2014 and Rosthern in 2017. Furthermore, RGA-G3Ap103 showed significant association at Milden in 2014. Based on association studies, RGA-G3Ap103 and PsC8780p118 may have some potential as markers for pea breeding.

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Molecular Fingerprinting and Hybridity Authentication in Cowpea Using Single Nucleotide Polymorphism Based Kompetitive Allele-Specific PCR Assay

Frontiers in Plant Science ◽

10.3389/fpls.2021.734117 ◽

2021 ◽

Vol 12 ◽

Author(s):

Patrick Obia Ongom ◽

Christian Fatokun ◽

Abou Togola ◽

Stella Salvo ◽

Oluwaseye Gideon Oyebode ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Principal Component ◽

Snp Markers ◽

Breeding Program ◽

Molecular Fingerprinting ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

Optimization of a breeding program for increased genetic gain requires quality assurance (QA) and quality control (QC) at key phases of the breeding process. One vital phase in a breeding program that requires QC and QA is the choice of parents and successful hybridizations to combine parental attributes and create variations. The objective of this study was to determine parental diversity and confirm hybridity of cowpea F1 progenies using KASP (Kompetitive Allele-Specific PCR)-based single nucleotide polymorphism (SNP) markers. A total of 1,436 F1 plants were derived from crossing 220 cowpea breeding lines and landraces to 2 elite sister lines IT99K-573-1-1 and IT99K-573-2-1 as male parents, constituting 225 cross combinations. The progenies and the parents were genotyped with 17 QC SNP markers via high-throughput KASP genotyping assay. The QC markers differentiated the parents with mean efficiency of 37.90% and a range of 3.4–82.8%, revealing unique fingerprints of the parents. Neighbor-Joining cladogram divided the 222 parents into 3 clusters. Genetic distances between parents ranged from 0 to 3.74 with a mean of 2.41. Principal component analysis (PCA) depicted a considerable overlap between parents and F1 progenies with more scatters among parents than the F1s. The differentiation among parents and F1s was best contributed to by 82% of the markers. As expected, parents and F1s showed a significant contrast in proportion of heterozygous individuals, with mean values of 0.02 and 0.32, respectively. KASP markers detected true hybridity with 100% success rate in 72% of the populations. Overall, 79% of the putative F1 plants were true hybrids, 14% were selfed plants, and 7% were undetermined due to missing data and lack of marker polymorphism between parents. The study demonstrated an effective application of KASP-based SNP assay in fingerprinting, confirmation of hybridity, and early detection of false F1 plants. The results further uncovered the need to deploy markers as a QC step in a breeding program.

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Constrution of a genetic map of rils derived from wheat (T. aestivum L.) varieties pamyatii azieva x paragon using high-throughput snp genotyping platform kasp - competative allele specific PCR

10.1101/740324 ◽

2019 ◽

Author(s):

K Yermekbayev ◽

S Griffiths ◽

M Chettry ◽

M Liverington-Waite ◽

S Orford ◽

...

Keyword(s):

Plant Breeding ◽

Genetic Map ◽

Dna Markers ◽

Mapping Population ◽

Snp Markers ◽

Linkage Groups ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr ◽

Segregating Population

AbstractThe main purposes of the study were i) to develop a first mapping population for bread wheat grown in Kazakhstan, ii) to construct its genetic map for further identification of genes associated with important agronomic traits.To the best of our knowledge this is the first segregating population and genetic map developed for Kazakh bread wheat. The work is an example of how plant breeding programs in Kazakhstan have started successfully deploying next generation plant breeding methods.The KASP (Compatative Allele Specific PCR) technology of LGC Group and SNP DNA-markers have been exploited to genotype and build a genetic map of the segregating population. The total length of the map was 1376 cM. A total 157 out of initial 178 SNP markers used formed 26 linkage groups leaving 1 duplicated and 20 unassigned markers. The threshold distance between markers was set ≤ 30 cM. Therefore, two linkage groups were obtained for chromosomes such as 2A, 2B, 2D, 3A, 5A, 6B and 7A. Despite one duplicated and 20 unassigned markers, the 157 KASP SNP markers that were mapped spanned A, B and D genomes of wheat. Kosambi Mapping function was employed to calculate recombination units between makers. RILs were developed through SSD method up to F4 generation. Almost 97% of identified alleles were useful in evaluating the population’s genetic diversity; the remaining 3% showed no outcome. As a result, 77 DNA markers were mapped for A, 74 for B and 27 for D genomes. The mapping population will be genotyped using high marker density array planform such as Illumina iSelect to obtain a genetic map with a relatively high coverage. Then, the population and high-resolution genetic map will be used to identify genes influencing wheat adaptation in Kazakhstan.

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Identification of Stripe Rust Resistance in Ethiopian Durum Wheat by Phenotypic Screening and Kompetitive Allele Specific PCR (KASP) SNP Markers

10.35248/2157-7471.19.10.483 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 1

Author(s):

Sisay Kidane Alemu ◽

Ayele Badebo ◽

Kassahun Tesfaye ◽

Cristobal Uauy

Keyword(s):

Durum Wheat ◽

Stripe Rust ◽

Rust Resistance ◽

Snp Markers ◽

Stripe Rust Resistance ◽

Phenotypic Screening ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

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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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MUPrimer : a tool for finding allele specific PCR-primers for homologous gene sequences

10.32469/10355/6660 ◽

2009 ◽

Author(s):

M. Mursaleen Ahmad

Keyword(s):

Pcr Primers ◽

Homologous Gene ◽

Gene Sequences ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

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

Agronomy ◽

10.3390/agronomy11050982 ◽

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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