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.

scholarly journals Transcriptome sequencing assisted discovery and computational analysis of novel SNPs associated with flowering in Raphanus sativus in-bred lines for marker-assisted backcross breeding

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Jinhee Kim ◽  
Abinaya Manivannan ◽  
Do-Sun Kim ◽  
Eun-Su Lee ◽  
Hye-Eun Lee
Keyword(s):  
Genetic Selection ◽  
Inbred Lines ◽  
Snp Markers ◽  
Functional Annotations ◽  
Specific Pcr ◽  
Polymorphic Snps ◽  
Allele Specific ◽  
Seed Purity Test ◽  
Backcross Breeding ◽  
Marker Assisted Backcross Breeding

Abstract The sequencing of radish genome aids in the better understanding and tailoring of traits associated with economic importance. In order to accelerate the genomics assisted breeding and genetic selection, transcriptomes of 33 radish inbred lines with diverse traits were sequenced for the development of single nucleotide polymorphic (SNP) markers. The sequence reads ranged from 2,560,543,741 bp to 20,039,688,139 bp with the GC (%) of 47.80–49.34 and phred quality score (Q30) of 96.47–97.54%. A total of 4951 polymorphic SNPs were identified among the accessions after stringent filtering and 298 SNPs with efficient marker assisted backcross breeding (MAB) markers were generated from the polymorphic SNPs. Further, functional annotations of SNPs revealed the effects and importance of the SNPs identified in the flowering process. The SNPs were predominantly associated with the four major flowering related transcription factors such as MYB, MADS box (AG), AP2/EREB, and bHLH. In addition, SNPs in the vital flowering integrator gene (FT) and floral repressors (EMBRYONIC FLOWER 1, 2, and FRIGIDA) were identified among the radish inbred lines. Further, 50 SNPs were randomly selected from 298 SNPs and validated using Kompetitive Allele Specific PCR genotyping system (KASP) in 102 radish inbred lines. The homozygosity of the inbred lines varied from 56 to 96% and the phylogenetic analysis resulted in the clustering of inbred lines into three subgroups. Taken together, the SNP markers identified in the present study can be utilized for the discrimination, seed purity test, and adjusting parental combinations for breeding in radish.

Validation of SNP markers associated with ascochyta blight resistance in pea

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.

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

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.

scholarly journals Comparative Assessment of Effectiveness of Alternative Genotyping Assays for Characterizing Carotenoids Accumulation in Tropical Maize Inbred Lines

Agronomy ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 2022
Author(s):  
Abdoul-Raouf Sayadi Maazou ◽  
Melaku Gedil ◽  
Victor O. Adetimirin ◽  
Silvestro Meseka ◽  
Wende Mengesha ◽  
...  
Keyword(s):  
Vitamin A Deficiency ◽  
Inbred Lines ◽  
Provitamin A ◽  
Carotenoid Content ◽  
Functional Markers ◽  
Favorable Alleles ◽  
Specific Pcr ◽  
Maize Inbred Lines ◽  
Maize Varieties ◽  
Allele Specific

The development of maize varieties with increased concentration of Provitamin A (PVA) is an effective and affordable strategy to combat vitamin A deficiency in developing nations. However, the considerably high cost of carotene analysis poses a major challenge for maize PVA biofortification, prompting the use of marker-assisted selection. Presently, two types of genotyping with PVA trait-linked functional markers have been developed and extensively used in breeding programs. The two systems are low throughput gel-based genotyping and genotyping with Kompetitive Allele-Specific PCR (KASP) single nucleotide polymorphism (SNPs) markers. Although the KASP SNPs genotyping was developed to replace the gel-based genotyping, studies have not been conducted to compare the effectiveness of the KASP SNPs markers with the gel-based markers. This study was conducted to assess the carotenoid content of 64 tropical PVA biofortified maize inbred lines containing temperate germplasm in their genetic backgrounds and screen them with both gel-based and KASP markers of PSY1, LCYE and crtRB1 genes. Many of the 64 inbred lines had PVA concentrations surpassing the 15 µg/g provitamin A breeding target set by the HarvestPlus Challenge Program. Favorable alleles of crtRB1, crtRB1 and the KASP SNPs markers were detected in 25 inbred lines with high PVA concentrations. Inbred lines with the favorable alleles of LCYE had the highest concentrations of non-PVA carotenoids, whereas those with the favorable alleles of crtRB1 had high levels of PVA carotenoids. Data from the sequenced region of LCYE revealed one SNP in the first intron that clearly differentiated the high and low β-carotene maize inbred lines. The results of our study demonstrate that the automated KASP SNPs markers can replace the gel-based genotyping for screening a large number of early generation maize inbred lines for PVA content.

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

2013 ◽  
Vol 1 (4) ◽  
pp. 1200406 ◽  
Author(s):  
Andres Garcia-Lor ◽  
Gema Ancillo ◽  
Luis Navarro ◽  
Patrick Ollitrault
Keyword(s):  
Snp Markers ◽  
Specific Pcr ◽  
Allele Specific ◽  
Allele Specific Pcr

Chromosome location and allele-specific PCR markers for marker-assisted selection of the oat crown rust resistance gene Pc91

2013 ◽  
Vol 32 (3) ◽  
pp. 679-686 ◽  
Author(s):  
B. N. Gnanesh ◽  
J. Mitchell Fetch ◽  
J. G. Menzies ◽  
A. D. Beattie ◽  
P. E. Eckstein ◽  
...  
Keyword(s):  
Resistance Gene ◽  
Marker Assisted Selection ◽  
Rust Resistance ◽  
Chromosome Location ◽  
Pcr Markers ◽  
Specific Pcr ◽  
Oat Crown Rust ◽  
Allele Specific ◽  
Allele Specific Pcr ◽  
Selection Of

scholarly journals 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

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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