scholarly journals EST-SNPs in bread wheat: discovery, validation, genotyping and haplotype structure

2009 ◽  
Vol 45 (No. 3) ◽  
pp. 106-116 ◽  
Author(s):  
S. Rustgi ◽  
R. Bandopadhyay ◽  
H.S. Balyan ◽  
P.K. Gupta
Keyword(s):  
Bread Wheat ◽  
In Silico Analysis ◽  
Nucleotide Polymorphisms ◽  
Specific Primers ◽  
Wheat Genotypes ◽  
Map Construction ◽  
Pcr Products ◽  
Allele Specific ◽  
Homoeologous Relationships

The present study involves discovery, validation and use of single-nucleotide polymorphisms (SNPs) in bread wheat utilizing 48 EST-contigs (individual contigs having 20-89 ESTs, derived from 2 to 11 different genotypes). In order to avoid a problem due to homoeologous relationships, the ESTs in each contig were classified into 175 sub-contigs (3.7 sub-contigs/EST-contig) using characteristic homoeologue sequence variants (HSVs), which had a density of 1 HSV every 136.7 bp. In silico analysis of sub-contigs led to the discovery of 230 candidate EST-SNPs with a density of 1SNP/273.9 bp. Locus specific primers (each primer pair flanking 1–18 SNPs) were designed utilizing one sub-contig each from 42 EST-contigs that contained SNPs, the remaining 6 contigs having no SNPs. To provide locus specificity to the PCR products, each primer was tagged with an HSV at its 3' end. Only 10 primer pairs, which gave each a characteristic solitary band, were utilized to validate EST-SNPs over 30 diverse bread wheat genotypes; 7 SNPs were validated through resequencing the PCR products. Allele specific primers were designed and utilized for genotyping of 50 diverse bread wheat accessions (including 30 bread wheat genotypes previously used for validation of SNPs), with an aim to test their utility in genotyping and map construction. The allele specific primers allowed the classification of 50 genotypes in two alternative allele groups for each SNP as expected, thus suggesting their utility for genotyping. Of the above 7 validated SNPs, 4 belonged to a solitary locus (PKS37); 7 haplotypes were available at this locus. Altogether, the results suggested that EST-SNPs constitute an important source of molecular markers for studies on wheat genomics.

scholarly journals Fine Mapping of qd1, a Dominant Gene that Regulates Stem Elongation in Bread Wheat

2021 ◽  
Vol 12 ◽  
Author(s):  
Yongdun Xie ◽  
Weiwei Zeng ◽  
Chaojie Wang ◽  
Daxing Xu ◽  
Huijun Guo ◽  
...  
Keyword(s):  
Bread Wheat ◽  
Stem Elongation ◽  
Dominant Gene ◽  
Nucleotide Polymorphisms ◽  
Sequencing Data ◽  
Specific Pcr ◽  
Yield Determination ◽  
Allele Specific ◽  
Stem Development ◽  
Kasp Markers

Stem elongation is a critical phase for yield determination and, as a major trait, is targeted for manipulation for improvement in bread wheat (Triticum aestivum L.). In a previous study, we characterized a mutant showing rapid stem elongation but with no effect on plant height at maturity. The present study aimed to finely map the underlying mutated gene, qd1, in this mutant. By analyzing an F2 segregating population consisting of 606 individuals, we found that the qd1 gene behaved in a dominant manner. Moreover, by using the bulked segregant RNA sequencing (BSR-seq)-based linkage analysis method, we initially mapped the qd1 gene to a 13.55 Mb region on chromosome 4B (from 15.41 to 28.96 Mb). This result was further confirmed in F2 and BC3F2 segregating populations. Furthermore, by using transcriptome sequencing data, we developed 14 Kompetitive Allele-Specific PCR (KASP) markers and then mapped the qd1 gene to a smaller and more precise 5.08 Mb interval from 26.80 to 31.88 Mb. To develop additional markers to finely map the qd1 gene, a total of 4,481 single-nucleotide polymorphisms (SNPs) within the 5.08 Mb interval were screened, and 25 KASP markers were developed based on 10x-depth genome resequencing data from both wild-type (WT) and mutant plants. The qd1 gene was finally mapped to a 1.33 Mb interval from 28.86 to 30.19 Mb on chromosome 4B. Four candidate genes were identified in this region. Among them, the expression pattern of only TraesCS4B02G042300 in the stems was concurrent with the stem development of the mutant and WT. The qd1 gene could be used in conjunction with molecular markers to manipulate stem development in the future.

scholarly journals In silico analysis of the fragrance gene (badh2) in Asian rice (Oryza sativa L.) germplasm and validation of allele specific markers

2020 ◽  
Vol 18 (2) ◽  
pp. 71-80
Author(s):  
Withanage Vidyani Erandika Withana ◽  
Rathanyaka Maudiyanselage Ramesha Eshani Kularathna ◽  
Nisha Sualri Kottearachchi ◽  
Deepthika S. Kekulandara ◽  
Jagath Weerasena ◽  
...  
Keyword(s):  
Oryza Sativa ◽  
In Silico ◽  
Oryza Sativa L ◽  
Major Gene ◽  
In Silico Analysis ◽  
Betaine Aldehyde Dehydrogenase ◽  
Nucleotide Polymorphisms ◽  
Rice Varieties ◽  
Asian Rice ◽  
Allele Specific

AbstractBadh2 of rice is considered to be the major gene responsible for the fragrance in rice. The wild type badh2 allele encodes betaine aldehyde dehydrogenase 2 (BADH2) enzyme while the mutated version of badh2 gene encodes non-functional BADH2 enzyme that leads to the accumulation of 2-acetyl-1-pyrroline (2AP), the principal fragrant compound in rice. There are many mutated recessive alleles causing fragrance in global rice germplasm, although the badh2.1 allele present in Basmati type rice is the most well-known among breeders. In this study, we attempted to reveal potential fragrance causing mutations, and the respective varieties carrying them, through in silico analysis based on the sequences available in the Rice SNP-Seek-Database of International Rice Research Institute. The sequences of 1878 rice accessions from 22 countries were analysed to identify mutations in each exon of badh2 comparatively with the non-fragrant ‘wildtype’ GenBank sequence in Nanjing11, Oryza sativa indica (EU770319.1). Results revealed that 63 varieties from 12 countries possessed the most prevalent allele, badh2.1 having an 8 bp deletion and three single nucleotide polymorphisms in the 7th exon. The second most prevalent allele in genotypes from Asia was badh2.7 having a ‘G’ insertion in the 14th exon. A novel allele with a T deletion in 9th exon was detected in a Thai rice accession. Rice varieties containing either badh2.1 or badh2.7 alleles could be identified with DNA markers for badh2.1 (frg) and badh2.7 (Bad2.7CAPS). The marker, Bad2.7CAPS, co-segregated with the fragrance phenotype in two crosses, confirming the possibility of employing it in marker assisted breeding.

scholarly journals Analysis of Clinically Relevant Single-Nucleotide Polymorphisms by Use of Microelectronic Array Technology

2002 ◽  
Vol 48 (12) ◽  
pp. 2124-2130 ◽  
Author(s):  
Rosa Santacroce ◽  
Antonia Ratti ◽  
Francesco Caroli ◽  
Barbara Foglieni ◽  
Alessandro Ferraris ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Factor Vii ◽  
Restriction Enzyme Digestion ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Ret Protooncogene ◽  
Mismatched Dna ◽  
Specific Pcr ◽  
Pcr Products ◽  
Allele Specific

Abstract Background: Microelectronic DNA chip devices represent an emerging technology for genotyping. We developed methods for detection of single-nucleotide polymorphisms (SNPs) in clinically relevant genes. Methods: Primer pairs, with one containing a 5′-biotin group, were used to PCR-amplify the region encompassing the SNP to be interrogated. After denaturation, the biotinylated strand was electronically targeted to discrete sites on streptavidin-coated gel pads surfaces by use of a Nanogen Molecular Workstation. Allele-specific dye-labeled oligonucleotide reporters were used for detection of wild-type and variant sequences. Methods were developed for SNPs in genes, including factor VII, β-globin, and the RET protooncogene. We genotyped 331 samples for five DNA variations in the factor VII gene, >600 samples from patients with β-thalassemia, and 15 samples for mutations within the RET protooncogene. All samples were previously typed by various methods, including DNA sequence analysis, allele-specific PCR, and/or restriction enzyme digestion of PCR products. Results: Analysis of amplified DNA required 4–6 h. After mismatched DNA was removed, signal-to-noise ratios were >5. More than 940 samples were typed with the microelectronic array platform, and results were totally concordant with results obtained previously by other genotyping methods. Conclusions: The described protocols detect SNPs of clinical interest with results comparable to those of other genotyping methods.

A novel technique for detecting single nucleotide polymorphisms by analyzing consumed allele-specific primers

2001 ◽  
Vol 22 (3) ◽  
pp. 418-420 ◽  
Author(s):  
Gotaro Watanabe ◽  
Kazuo Umetsu ◽  
Isao Yuasa ◽  
Michihiko Sato ◽  
Munechika Sakabe ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Nucleotide Polymorphisms ◽  
Specific Primers ◽  
Single Nucleotide ◽  
Novel Technique ◽  
Allele Specific

scholarly journals THE LINE RICO IS THE EARLIEST MATURING ACCESSION IN THE VIR COLLECTION OF SPRING BREAD WHEAT

2020 ◽  
Vol 180 (4) ◽  
pp. 94-98
Author(s):  
B. V. Rigin ◽  
E. V. Zuev ◽  
A. S. Andreeva ◽  
Z. S. Pyzhenkova ◽  
I. I. Matvienko
Keyword(s):  
Bread Wheat ◽  
Wheat Breeding ◽  
Development Rate ◽  
Wheat Cultivars ◽  
Specific Primers ◽  
Spring Bread Wheat ◽  
F2 Population ◽  
Plant Sensitivity ◽  
Allele Specific ◽  
The Difference

Background. To optimize the process of bread wheat breeding for earliness and environmental adaptability, searching for new source material is a crucial task. The ultra-early line Rico (k-65588) – Triticum aestivum var. erythrospermum Koern. – stands out among the bread wheat accessions from the VIR collection for its important adaptive features.Materials and methods. Spring wheat accessions with different speed of development were selected from the VIR collection for this study, along with the ultra-early lines Rifor 1 ... 10 (F6-7 Rico × Forlani Roberto k-42641) and Fori 1 ... 8 (k-65589 ... k-65596) (F4 Foton k-55696 × Rico). Their responses to a short 12-hour day were assessed. Vernalization conditions were 30 days at 3°C. The genetics of plant sensitivity to vernalization and photoperiods was studied using allele-specific primers for the genes Vrn-A1, Vrn-B1, Vrn-D1 and Ppd-D1.Results and conclusions. In the Northwest of Russia, the period from seeding to heading for Rico plants was 39.9 ± 1.49 days, or 14.8 ± 1.22 days less than for the released commercial wheat cultivars. Among the 8400 wheat accessions, studied by the Wheat Genetic Resources Department of VIR in this area from 1948 to 2018, the shortest period from germination to heading was observed in the line Rico: 29 (28–30) days. The absence of response to vernalization in Rico, Fori and Rifor lines was determined by the dominant alleles Vrn-A1, Vrn-B1 and Vrn-D1. Photoperiodism in Rico and partially in Rifor was controlled by at least two genes: Ppd-D1 and Ppd-B1. In the F2 population of Rico hybrids with 8 wheat accessions no transgression was observed beyond the limits of Rico‘s variation. The difference in the development rate between Rico and other wheat accessions is controlled by two or three non-allelic genes. Rifor lines can compete in productivity with commercialized wheat cultivars.

scholarly journals HRM technology for the identification and characterization of INDEL and SNPs mutations in genes involved in drought and salt tolerance of durum wheat

2011 ◽  
Vol 9 (2) ◽  
pp. 166-169 ◽  
Author(s):  
Linda Mondini ◽  
Miloudi M. Nachit ◽  
Enrico Porceddu ◽  
Mario A. Pagnotta
Keyword(s):  
Durum Wheat ◽  
Abiotic Stress Tolerance ◽  
Nucleotide Polymorphisms ◽  
Specific Primers ◽  
Single Nucleotide ◽  
Drought And Salt Tolerance ◽  
Pcr Products ◽  
Identification And Characterization ◽  
Single Snps

WRKY transcription factors are one of the largest families of transcriptional regulators and form an integral part of signalling webs which modulate many plant processes, such as abiotic stress tolerance. In the present paper, an innovative method has been applied to identify novel WRKY-1 alleles involved in the responses to salt and drought stresses in Triticum durum. This technique involves scanning for sequencing variations in cDNA-derived PCR amplicons, using high-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM represents a novel advance in detection of single-nucleotide polymorphisms (SNPs) by measuring temperature-induced strand separation of short PCR amplicons. The use of this approach is still limited in the field of plant biology. Here, HRM analysis has been applied to the discovery and genotyping of durum wheat SNPs. Specific primers have been designed, starting at multi-alignment of WRKY-1-conserved portions. The PCR amplicons, containing single SNPs, produce distinctive HRM profiles, and by sequencing the PCR products identified, SNPs have been characterized and validated. The results showed that all the revealed SNPs are located on salt-tolerant varieties, confirming their value in breeding activities.

scholarly journals Application of Allele Specific Markers in Determining Alleles at VRN-1 Loci in Bread Wheat Genotypes

2019 ◽  
Vol 11 (32) ◽  
pp. 133-143
Author(s):  
Monireh Nazari ◽  
Khalil Zaynali Nezhad ◽  
Saeid Navabpour ◽  
Hassan Soltanlo ◽  
Mohamdhadi Pahlavani ◽  
...  
Keyword(s):  
Bread Wheat ◽  
Wheat Genotypes ◽  
Allele Specific

scholarly journals Haplotype-Contained PCR Products Analysis by Sequencing with Selective Restriction of Primer Extension

2017 ◽  
Vol 2017 ◽  
pp. 1-9
Author(s):  
Liu Wang ◽  
Pengfeng Xiao
Keyword(s):  
Sanger Sequencing ◽  
Haplotype Analysis ◽  
Read Length ◽  
Clinical Samples ◽  
Heterozygous Snps ◽  
Specific Primers ◽  
Products Analysis ◽  
Pcr Products ◽  
Allele Specific ◽  
Short Read Length

We develop a strategy for haplotype analysis of PCR products that contained two adjacent heterozygous loci using sequencing with specific primers, allele-specific primers, and ddNTP-blocked primers. To validate its feasibility, two sets of PCR products, including two adjacent heterozygous SNPs, UGT1A1⁎6 (rs4148323) and UGT1A1⁎28 (rs8175347), and two adjacent heterozygous SNPs, K1637K (rs11176013) and S1647T (rs11564148), were analyzed. Haplotypes of PCR products, including UGT1A1⁎6 and UGT1A1⁎28, were successfully analyzed by Sanger sequencing with allele-specific primers. Also, haplotypes of PCR products, including K1637K and S1647T, could not be determined by Sanger sequencing with allele-specific primers but were successfully analyzed by pyrosequencing with ddNTP-blocked primers. As a result, this method is able to effectively haplotype two adjacent heterozygous PCR products. It is simple, fast, and irrespective of short read length of pyrosequencing. Overall, we fully hope it will provide a new promising technology to identify haplotypes of conventional PCR products in clinical samples.

scholarly journals Warfarin Genotyping in a Single PCR Reaction for Microchip Electrophoresis

2012 ◽  
Vol 58 (4) ◽  
pp. 725-731 ◽  
Author(s):  
Brian L Poe ◽  
Doris M Haverstick ◽  
James P Landers
Keyword(s):  
Turnaround Time ◽  
Microchip Electrophoresis ◽  
Amplification Refractory Mutation System ◽  
Sample Population ◽  
Nucleotide Polymorphisms ◽  
Specific Pcr ◽  
Mutation System ◽  
Pcr Products ◽  
Allele Specific ◽  
Cytochrome P450 Family

Abstract BACKGROUND Warfarin is the most commonly prescribed oral anticoagulant medication but also is the second leading cause of emergency room visits for adverse drug reactions. Genetic testing for warfarin sensitivity may reduce hospitalization rates, but prospective genotyping is impeded in part by the turnaround time and costs of genotyping. Microfluidics-based assays can reduce reagent consumption and analysis time; however, no current assay has integrated multiplexed allele-specific PCR for warfarin genotyping with electrophoretic microfluidics hardware. Ideally, such an assay would use a single PCR reaction and, without further processing, a single microchip electrophoresis (ME) run to determine the 3 single-nucleotide polymorphisms (SNPs) affecting warfarin sensitivity [i.e., CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) *2, CYP2C9 *3, and the VKORC1 (vitamin K epoxide reductase complex 1) A/B haplotype]. METHODS We designed and optimized primers for a fully multiplexed assay to examine 3 biallelic SNPs with the tetraprimer amplification refractory mutation system (T-ARMS). The assay was developed with conventional PCR equipment and demonstrated for microfluidic infrared-mediated PCR. Genotypes were determined by ME on the basis of the pattern of PCR products. RESULTS Thirty-five samples of human genomic DNA were analyzed with this multiplex T-ARMS assay, and 100% of the genotype determinations agreed with the results obtained by other validated methods. The sample population included several genotypes conferring warfarin sensitivity, with both homozygous and heterozygous genotypes for each SNP. Total analysis times for the PCR and ME were approximately 75 min (1-sample run) and 90 min (12-sample run). CONCLUSIONS This multiplexed T-ARMS assay coupled with microfluidics hardware constitutes a promising avenue for an inexpensive and rapid platform for warfarin genotyping.

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