Functional molecular markers and high-resolution melting curve analysis of low phytic acid mutations for marker-assisted selection in rice

Tan, Yuanyuan; Fu, Haidong; Zhao, Haijun; Lu, Sha; Fu, Junjie; Li, Youfa; Hairui, Cui; Shu, Qingyao

doi:10.1007/s11032-012-9809-5

Cited by 29 publications

(21 citation statements)

References 42 publications

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“…Here we carried out genotyping the polymorphism in the loop structure through HRM approach, which has been commonly used in clinical chemistry, human pathology and patient genotyping since it was invented in 2003 1. It has been introduced into genotyping of crop plants including rice 17. Its advantage is that it is performed in a real-time PCR device, and the PCR amplification and HRM analysis are performed in a closed tube, and effectively single step18.…”

Section: Discussionmentioning

confidence: 99%

Loop Nucleotide Polymorphism in a Putative miRNA Precursor Associated with Seed Length in Rice (Oryza sativa L.)

Wang¹,

Ye²,

Tang³

et al. 2013

Int. J. Biol. Sci.

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The terminal loop region of primary miRNA transcripts is an important determinant controlling miRNA function in human, animals and plants. However, the effects of polymorphisms in loop region of miRNA precusors on phenotypic variation have not been reported, especially on agronomic traits in rice. From rice japonica variety Koshihikari and indica Guichao2, we isolated a precursor of putative miRNA osa-MIR2923a. We detected a GG/AA polymorphism in the loop structure from japonica and indica rice varieties, which was corresponding to japonica/indica rice differentiation. By using high-resolution melting (HRM) analysis, we measured the polymorphisms in a RIL (recombinant inbred lines) population derived from japonica variety Koshihikari and indica Guichao2. We found that the GG/AA polymorphism in the osa-MIR2923a loop was correlated to grain length and length-width ratio. We further found the significant association between seed length and GG/AA polymorphism in a population consisting of 72 rice landraces. Three targets were predicted, whose expressions showed significant differences between the two varieties. Our results suggested that the putative miRNA precursor and the three target genes could play functional roles for indica/japonica seed differentiation.

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Section: Discussionmentioning

confidence: 99%

Loop Nucleotide Polymorphism in a Putative miRNA Precursor Associated with Seed Length in Rice (Oryza sativa L.)

Wang¹,

Ye²,

Tang³

et al. 2013

Int. J. Biol. Sci.

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“…Genomic DNAs were extracted from 32 plants of each P 3 M 3 line and subjected to high-resolution melting (HRM) curve analysis, according to Tan et al (2013). In brief, PCRs were performed in a 10-µl volume with 25 ng of DNA, 5 µl of 2× master mix (TOYOBO Co., Ltd.), 0.2 µl each of 10 µmol/L primers, and 1 µl of 10× EvaGreen (Biotium, USA), covered with a drop of mineral oil.…”

Section: Verification Of Identified Mutationsmentioning

confidence: 99%

Frequency and type of inheritable mutations induced by γ rays in rice as revealed by whole genome sequencing

Zheng

Hairui

et al. 2016

J. Zhejiang Univ. Sci. B

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Mutation breeding is based on the induction of genetic variations; hence knowledge of the frequency and type of induced mutations is of paramount importance for the design and implementation of a mutation breeding program. Although γ ray irradiation has been widely used since the 1960s in the breeding of about 200 economically important plant species, molecular elucidation of its genetic effects has so far been achieved largely by analysis of target genes or genomic regions. In the present study, the whole genomes of six γ-irradiated M 2 rice plants were sequenced; a total of 144-188 million high-quality (Q>20) reads were generated for each M 2 plant, resulting in genome coverage of >45 times for each plant. Single base substitution (SBS) and short insertion/deletion (Indel) mutations were detected at the average frequency of 7.5×10 −6 -9.8×10 −6 in the six M 2 rice plants (SBS being about 4 times more frequent than Indels). Structural and copy number variations, though less frequent than SBS and Indel, were also identified and validated. The mutations were scattered in all genomic regions across 12 rice chromosomes without apparent hotspots. The present study is the first genome-wide single-nucleotide resolution study on the feature and frequency of γ irradiation-induced mutations in a seed propagated crop; the findings are of practical importance for mutation breeding of rice and other crop species.

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“…almond (Wu et al 2008), barley (Hofinger et al 2009), potato (Koeyer et al 2010), rice (Li et al 2011;Tan et al 2013), grapevine (Emanuelli et al 2014) and ryegrass (Birrer et al 2014). In the present study, we examined four approaches of HRM in genotyping loci underlying rice PGMS and TGMS: direct amplicon scanning, scanning of amplicons from test samples mixed with known genotype DNA (of mutant or wild type), scanning of amplicons with an oligonucleotide probe, and CADMA-based melt analysis.…”

Section: Discussionmentioning

confidence: 99%

“…However, it is also known that amplicon melting analysis may not work for some variants (Reed et al 2007). Hence additional procedures might be necessary, e.g., pre-PCR mixing of DNA templates with a known genotype-wild type (WT) or mutant type (MT) (Tan et al 2013), use of oligonucleotide probes (Montgomery et al 2007), or competitive amplification of differentially melting amplicons (CADMA), which was recently reported to be able to detect all mutation types in human samples (Kristensen et al 2012). The CADMA method employs a three-primer system: a mutation-specific primer that introduces one or more mutations to decrease the melting temperature in the mutation-containing amplicon, a second overlapping primer and a third common primer to amplify both the mutated and WT sequences.…”

Section: Electronic Supplementary Materialsmentioning

confidence: 99%

“…For standard HRM analysis, genomic DNAs were extracted from leaf tissues following a modified CTAB method (Tan et al 2013) and adjusted to a final concentration of *50 ng/lL after quantification using a Nano-Drop 2000 (Thermo Scientific, USA). For seed purity testing, the DNA was extracted from single seeds following Tan et al (2013).…”

Section: Dna Extraction Pcr and Hrm Analysismentioning

confidence: 99%

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Competitive amplification of differentially melting amplicons facilitates efficient genotyping of photoperiod- and temperature-sensitive genic male sterility in rice

et al. 2014

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Genic male sterility conditioned by either photoperiod (PGMS) or temperature (TGMS) is the key trait that has enabled the establishment of the two-line hybrid rice (Oryza sativa L.) system. The PGMS trait is known to result from a C?G mutation in a long noncoding RNA gene (lncR) and the TGMS from a premature stop codon mutation (TC/GC?TA) in an RNase Z gene (RNZ). To develop an efficient genotyping method capable of distinguishing all potential genotypes for the loci of lncR and RNZ, the present study explored the capacity of four approaches in high-resolution melting (HRM) curve analysis, viz. amplicon scanning, amplicon scanning with pre-PCR addition of mutant DNA template or inclusion of an unlabeled oligonucleotide probe, and melting analysis by competitive amplification of differentially melting amplicons (CADMA). The former three approaches were demonstrated to be able to differentiate only a few genotypes; in contrast, CADMA was shown to be able to discriminate all possible genotypes of both genes. For the lncR locus, HRM curves of three possible genotypes (PGMS, wild-type line and their hybrid) had peak fluorescence differences (DFs) of *0.075 to *0.20, significantly greater than the threshold ([0.05) for differentiation. For the RNZ locus, the five genotypes that would be of interest to breeders and seed technologists were separated according their HRM curves with DFs [ *0.1 between any two genotypes. The usefulness of the CADMA-based approach was further validated by analyzing a number of uncharacterized genotypes including P/TGMS lines, F1 hybrid seeds and F2 plants derived from crosses of P/TGMS lines with wild-type lines. Taken together, the present study proved the usefulness of CADMA in plant genotyping and established a CADMA-based HRM analysis method for high-throughput P/TGMS genotyping, which can be applied not only to markerassisted selection of PGMS and TGMS rice but also to seed purity testing in the two-line hybrid rice system. Electronic supplementary materialThe online version of this article (

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Functional molecular markers and high-resolution melting curve analysis of low phytic acid mutations for marker-assisted selection in rice

Cited by 29 publications

References 42 publications

Loop Nucleotide Polymorphism in a Putative miRNA Precursor Associated with Seed Length in Rice (Oryza sativa L.)

Loop Nucleotide Polymorphism in a Putative miRNA Precursor Associated with Seed Length in Rice (Oryza sativa L.)

Frequency and type of inheritable mutations induced by γ rays in rice as revealed by whole genome sequencing

Competitive amplification of differentially melting amplicons facilitates efficient genotyping of photoperiod- and temperature-sensitive genic male sterility in rice

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