Development of an HRM-based, safe and high-throughput genotyping system for two low phytic acid mutations in soybean

Tan, Yuanyuan; Yu, Xiaomin; Shu, Qingyao; Zhang, Huali; Wang, Shou-gen; Yuan, Fengjie; Shi, Chao

doi:10.1007/s11032-016-0529-0

Cited by 12 publications

(13 citation statements)

References 29 publications

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“…Following nested PCR, plates were analyzed in the Lightscanner instrument (Idaho Technology Inc., USA) and subjected to HRM analysis according to Tan et al (2016). Samples with relative fluorescence differences (| F|) >0.05 with respect to the reference (Hofinger et al, 2009) were considered to be putative mutants thus the entire amplicons of the gene were re-amplified from DNA of each such individuals for Sanger sequencing.…”

Section: Screening For Small Mutationsmentioning

confidence: 99%

CRISPR-Cas9 Multiplex Editing of the α-Amylase/Trypsin Inhibitor Genes to Reduce Allergen Proteins in Durum Wheat

Camerlengo

Frittelli

Sparks

et al. 2020

Front. Sustain. Food Syst.

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Wheat and its derived foods are widespread, representing one of the main food sources globally. During the last decades, the incidence of disorders related to wheat has become a global issue for the human population, probably linked to the spread of wheat-derived foods. It has been ascertained that structural and metabolic proteins, like α-amylase/trypsin inhibitors (ATI), are involved in the onset of wheat allergies (bakers' asthma) and probably Non-Coeliac Wheat Sensitivity (NCWS). The ATI are a group of exogenous protease inhibitors, which are encoded by a multigene family dispersed over several chromosomes in durum and bread wheat. WTAI-CM3 and WTAI-CM16 subunits are considered among the main proteins involved in the onset of bakers' asthma and probably NCWS. A CRISPR-Cas9 multiplexing strategy was used to edit the ATI subunits WTAI-CM3 and WTAI-CM16 in the grain of the Italian durum wheat cultivar Svevo with the aim to produce wheat lines with reduced amount of potential allergens involved in adverse reactions. Using a marker gene-free approach, whereby plants are regenerated without selection agents, homozygous mutant plants without the presence of CRISPR vectors were obtained directly from T 0 generation. This study demonstrates the capability of CRISPR technology to knock out immunogenic proteins in a reduced time compared to conventional breeding programmes. The editing of the two target genes was confirmed either at molecular (sequencing and gene expression study) or biochemical (immunologic test) level. Noteworthy, as a pleiotropic effect, is the activation of the ATI 0.28 pseudogene in the edited lines.

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Section: Screening For Small Mutationsmentioning

confidence: 99%

CRISPR-Cas9 Multiplex Editing of the α-Amylase/Trypsin Inhibitor Genes to Reduce Allergen Proteins in Durum Wheat

Camerlengo

Frittelli

Sparks

et al. 2020

Front. Sustain. Food Syst.

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“…Following PCR, plates were transferred to a Lightscanner (Idaho Technology Inc., USA) and subjected to HRM analysis according to Tan et al (2016). In brief, the temperature was ramped up from 55 to 95 °C at 0.1 °C per second and data were analyzed using its proprietary software, Call ITTM 2.0 (Idaho Technology Inc., USA) after normalization and temperature shifting of the melting curves according to the Lightscanner Operator's Manual (Idaho Technology Inc.).…”

Section: Hrm Identification Of Genetic Variationsmentioning

confidence: 99%

HRM-facilitated rapid identification and genotyping of mutations induced by CRISPR/Cas9 mutagenesis in rice

Liu

et al. 2018

Crop Breed. Appl. Biotechnol.

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The clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated (Cas) technology has recently emerged as a powerful genomic editing tool with great potential for crop breeding. However, commonly used protocols for screening of CRISPR/Cas9-induced mutations are laborious, timeconsuming, and costly. In the present study we examined the applicability of high resolution melting (HRM) analysis fast screening CRISPR/Cas9-induced mutations in T 0 plants and subsequent genotyping of T 1 populations. Comparative analysis demonstrated that HRM analysis could identify mutant T 0 plants carrying various types of mutation, including single nucleotide substitutions and short insertion/deletions, with false positive/negative rates of 0-2.78%. Furthermore, T 1 plants derived from single T 0 plants could be correctly genotyped by HRM analysis using WT parents and T 0 plants as controls. We hence recommend the adoption of HRM analysis in CRISPR/Cas9 mediated genetic studies and breeding in rice and other crop species. This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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“…To establish the HRM-TILLING and genotyping of M 3 plants, genomic DNA was extracted from leaf tissues using a modified cetyltrimethylammonium bromide (CTAB) method according to Li et al (2016) and adjusted to a final concentration of about 50 ng/µl after quantification using a Nanodrop 2000 (Thermo Scientific, USA). For mutation screening, DNA of M 2 seedlings was extracted using a simple, safe, and fast DNA extraction protocol adopted from Tan et al (2016). Briefly, leaf disks (diameter about 2 mm) were collected from samples of four M 2 seedlings using a hole puncher, and then mixed and extracted into 96-well polymerase chain reaction (PCR) plates.…”

Section: Dna Extractionmentioning

confidence: 99%

“…Following PCR, plates were transferred to a LightScanner (Idaho Technology Inc., USA) and subjected to HRM analysis according to Tan et al (2016). In brief, the temperature was ramped up from 55 to 95 °C at 0.1 °C/s, and data were analyzed using the proprietary software, Call ITTM 2.0 (Idaho Technology Inc., USA) after normalization and temperature shifting of the melting curves according to the LightScanner Operator's Manual (Idaho Technology Inc.).…”

Section: Hrm Analysismentioning

confidence: 99%

High-resolution melting-based TILLING of γ ray-induced mutations in rice

Liu

Fu³

et al. 2018

J. Zhejiang Univ. Sci. B

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Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics strategy for the high-throughput screening of induced mutations. γ radiation, which often induces both insertion/deletion (Indel) and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting (HRM) analysis. Pooled rice (Oryza sativa) samples mixed at a 1:7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M plants is recommended for screening γ ray-induced mutants in rice. For demonstration, a γ ray-mutagenized M rice population (n=4560) was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution (G→A) and one single nucleotide insertion (A) were identified in OsLCT1, and one trinucleotide (TTC) deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ ray mutagenesis to the breeding of rice and other seed crops.

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Development of an HRM-based, safe and high-throughput genotyping system for two low phytic acid mutations in soybean

Cited by 12 publications

References 29 publications

CRISPR-Cas9 Multiplex Editing of the α-Amylase/Trypsin Inhibitor Genes to Reduce Allergen Proteins in Durum Wheat

CRISPR-Cas9 Multiplex Editing of the α-Amylase/Trypsin Inhibitor Genes to Reduce Allergen Proteins in Durum Wheat

HRM-facilitated rapid identification and genotyping of mutations induced by CRISPR/Cas9 mutagenesis in rice

High-resolution melting-based TILLING of γ ray-induced mutations in rice

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