Development and validation of high-throughput and low-cost STARP assays for genes underpinning economically important traits in wheat

Wu, Yifan; Li, Ming; He, Zhonghu; Dreisigacker, Susanne; Wen, Weie; Jin, Hui; Zhai, Shengnan; Li, Faji; Gao, Fengjie; Liu, Jindong; Wang, Rongge; Zhang, Pingzhi; Wan, Yat-wah; Cao, Shuanghe; Xia, Xianchun

doi:10.1007/s00122-020-03609-w

Cited by 12 publications

(6 citation statements)

References 92 publications

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“…Many SNP genotyping methods, especially TaqMan and Molecular Beacons, where allele-specific and dual-labeled parts are combined in a single molecular probe, are too expensive to be adopted by smaller laboratories (Edenberg and Liu, 2009 ; Kumar et al, 2012 ; You et al, 2018 ; Zhao et al, 2019 ; Zhu et al, 2021 ). The latest generation of SNP genotyping methods, for example, Amplifluor, KASP, rhAmp, and STARP, use universal molecular probes which are not related to any particular SNP and, therefore, can be applied to different SNP analyses (Rasheed et al, 2016 ; Long et al, 2017 ; Broccanello et al, 2018 ; Ayalew et al, 2019 ; Li et al, 2019 ; Wu et al, 2020 ). Such SNP technologies are significantly cheaper than the previous methods, but they still remain relatively expensive for laboratories with a limited budget.…”

Section: Discussionmentioning

confidence: 99%

“…Other ASP-UP methods, such as Amplifluor-like and similar, are less expensive since all methods are unrestricted and published (Rickert et al, 2004 ; Rasheed et al, 2017 ; Zotova et al, 2019 , 2020 ). The recently reported STARP or STAR-PCR method is similar but with different sequences for the length and shape of the “stem-loop” in the “hair-pin” of their UPs (Rasheed et al, 2016 ; Long et al, 2017 ; Li et al, 2019 ; Wu et al, 2020 ). In contrast, another SNP genotyping method, “rhAmp,” is based on the use of H2-dependent RNase together with UPs and ASPs (Integrated DNA Technologies, USA).…”

Section: Introductionmentioning

confidence: 99%

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Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET

et al. 2022

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The proposed method is a modified and improved version of the existing “Allele-specific q-PCR” (ASQ) method for genotyping of single nucleotide polymorphism (SNP) based on fluorescence resonance energy transfer (FRET). This method is similar to frequently used techniques like Amplifluor and Kompetitive allele specific PCR (KASP), as well as others employing common universal probes (UPs) for SNP analyses. In the proposed ASQ method, the fluorophores and quencher are located in separate complementary oligonucleotides. The ASQ method is based on the simultaneous presence in PCR of the following two components: an allele-specific mixture (allele-specific and common primers) and a template-independent detector mixture that contains two or more (up to four) universal probes (UP-1 to 4) and a single universal quencher oligonucleotide (Uni-Q). The SNP site is positioned preferably at a penultimate base in each allele-specific primer, which increases the reaction specificity and allele discrimination. The proposed ASQ method is advanced in providing a very clear and effective measurement of the fluorescence emitted, with very low signal background-noise, and simple procedures convenient for customized modifications and adjustments. Importantly, this ASQ method is estimated as two- to ten-fold cheaper than Amplifluor and KASP, and much cheaper than all those methods that rely on dual-labeled probes without universal components, like TaqMan and Molecular Beacons. Results for SNP genotyping in the barley genes HvSAP16 and HvSAP8, in which stress-associated proteins are controlled, are presented as proven and validated examples. This method is suitable for bi-allelic uniplex reactions but it can potentially be used for 3- or 4-allelic variants or different SNPs in a multiplex format in a range of applications including medical, forensic, or others involving SNP genotyping.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET

et al. 2022

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“…Compared with neutral DNA markers, FMs have the advantage of being entirely associated with target genes. Therefore, FMs have been preferred in biodiversity studies (van Tienderen et al 2002;Wu et al 2020).…”

Section: Discussionmentioning

confidence: 99%

Genetic Characterization of Rainfed Durum Wheat Genotypes Based On Functional Markers Associated With Grain Quality Proteins

Kheibary

Azizinezhad

Mehrabi

et al. 2021

Preprint

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Analysis of genetic diversity provides helpful information necessary to develop the breeding and conservation strategies of crops. In this study, the genetic diversity and population structure of 90 durum wheat genotypes maintained at Sararud Dryland Agricultural Research Institute, Kermanshah, Iran, were evaluated by using 23 gene-specific markers (functional markers, FMs) encoding high and low molecular weight glutenin and gliadin alleles. Results showed that 12 out of the 23 FMs used were polymorphic and amplified 52 polymorphic loci. Primer Ax2 ⃰ had the highest discriminatory power. The population structure analysis classified the durum wheat collection into four populations. On average, population 4, consisting of 8 genotypes, had the highest allele number as well as genetic variation. Analysis of molecular variance indicated that 82% of the total variation was distributed among populations. The diversity among populations and gene flow were 0.14 and 3.03, respectively. The Jaccard distance coefficient revealed that genetic dissimilarities ranged from 0.031 between G62 and G65 to 0.725 between G36 and G51. Neighbor-joining method clustered individuals into six main groups. Results showed a remarkable level of genetic diversity among studied durum wheat genotypes which can be of interest for future breeding programs.

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“…In recent years, with the release of the wheat and closely related species genome sequence, and numerous transcriptome datasets (Duan et al, 2012;Kumar et al, 2015;Yang et al, 2022), all of these might lead to greater convenience for gene mapping, discovery of candidate genes, gene cloning, and development of markers, especially in the area of marker development, such as simple sequence repeat (SSR) markers, cleaved amplified polymorphic sequence (CAPS) markers, kompetitive allelespecific PCR (KASP) markers, and semi-thermal asymmetric reverse PCR (STARP) markers (Wu et al, 2020). The rapid evolution of molecular technology has provided powerful tools to dissect complex traits ; many molecular markers for kernel traits have been developed, especially in KASP markers, for example, KASP-AX-111112626 (tightly linked to kernel length QTL QKL.sicau-AM-3B), KASP-AX-108974756 (tightly linked to kernel width QTL QKW.sicau-AM-4B) (Zhou et al, 2021), and KASP-AX-109379070 (tightly linked to kernel length QTL QKL.sicau-2SY-1B) (Qu et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

QTL cluster analysis and marker development for kernel traits based on DArT markers in spring bread wheat (Triticum aestivum L.)

Zeng

Zhao²,

Wang³

et al. 2023

Front. Plant Sci.

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Genetic dissection of yield component traits including kernel characteristics is essential for the continuous improvement in wheat yield. In the present study, one recombinant inbred line (RIL) F6 population derived from a cross between Avocet and Chilero was used to evaluate the phenotypes of kernel traits of thousand-kernel weight (TKW), kernel length (KL), and kernel width (KW) in four environments at three experimental stations during the 2018–2020 wheat growing seasons. The high-density genetic linkage map was constructed with the diversity arrays technology (DArT) markers and the inclusive composite interval mapping (ICIM) method to identify the quantitative trait loci (QTLs) for TKW, KL, and KW. A total of 48 QTLs for three traits were identified in the RIL population on the 21 chromosomes besides 2A, 4D, and 5B, accounting for 3.00%–33.85% of the phenotypic variances. Based on the physical positions of each QTL, nine stable QTL clusters were identified in the RILs, and among these QTL clusters, TaTKW-1A was tightly linked to the DArT marker interval 3950546–1213099, explaining 10.31%–33.85% of the phenotypic variances. A total of 347 high-confidence genes were identified in a 34.74-Mb physical interval. TraesCS1A02G045300 and TraesCS1A02G058400 were among the putative candidate genes associated with kernel traits, and they were expressed during grain development. Moreover, we also developed high-throughput kompetitive allele-specific PCR (KASP) markers of TaTKW-1A, validated in a natural population of 114 wheat varieties. The study provides a basis for cloning the functional genes underlying the QTL for kernel traits and a practical and accurate marker for molecular breeding.

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Development and validation of high-throughput and low-cost STARP assays for genes underpinning economically important traits in wheat

Cited by 12 publications

References 92 publications

Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET

Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET

Genetic Characterization of Rainfed Durum Wheat Genotypes Based On Functional Markers Associated With Grain Quality Proteins

QTL cluster analysis and marker development for kernel traits based on DArT markers in spring bread wheat (Triticum aestivum L.)

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