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

High-resolution melting-based TILLING of γ ray-induced mutations in rice 基于高分辨率熔解曲线技术的水稻伽玛射线诱发突变的TILLIN体系 1 3 first-author 1 4 3 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:qyshu@zju.edu.cn †E-mail:qyshu@zju.edu.cn qyshu@zju.edu.cn National Key Laboratory of Rice Biology, Institute of Crop Sciences, Zhejiang University, Hangzhou 310058, China National Key Laboratory of Rice Biology, Institute of Crop Sciences, Zhejiang University, Hangzhou 310058, China Hubei Collaborative Innovation Center for Grain Industry, Jingzhou 434025, China Hubei Collaborative Innovation Center for Grain Industry, Jingzhou 434025, China Institute of Nuclear Agricultural Sciences, Zhejiang University, Hangzhou 310029, China Institute of Nuclear Agricultural Sciences, Zhejiang University, Hangzhou 310029, China Jiaxing Academy of Agricultural Sciences, Jiaxing 314016, China Jiaxing Academy of Agricultural Sciences, Jiaxing 314016, China 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 2 plants is recommended for screening γ ray-induced mutants in rice. For demonstration, a γ ray-mutagenized M 2 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. 目的 2 建立适用于筛选伽马射线诱发突变的、基于高分辨率熔解曲线(high-resolution melting,HRM)技术的高通量定向诱导基因组局部突变技术(Targeting Induced Local Lesions IN Genomes,TILLING)体系. 创新点 2 建立起了基于HRM技术、适用于伽玛射线诱发的小片段插入/缺失突变的高通量TILLING体系(HRM-TILLING). 方法 2 通过不同野生型/突变型比例混池DNA的HRM分析,确定HRM检测不同类型插入/缺失突变的能力,确定M 2 植株突变检测的适宜混池比例,并用一个伽玛诱变M 2 群体( n =4560)筛选 OsLCT1 和 SPDT 两个基因的突变体,确定实际效果. 结论 2 以4株M 2 植株混样,采用HRM可以有效检出突变.建立的基于HRM的TILLING体系适用于伽玛射线诱发突变的高通量筛选. Mutation screening High-resolution melting (HRM) analysis Targeting Induced Local Lesions IN Genomes (TILLING) Mutant γ ray Rice 突变筛选 高分辨率熔解曲线(HRM) 定向诱导基因组局部突变技术(TILLING) 突变体 插入缺失 伽玛射线 水稻 Introduction 1 Mutants are an important genetic resource for both functional genomics and crop breeding. For the fast and efficient screening of induced mutations, McCallum et al. ( 2000 2000 ) developed a novel system, now known as TILLING (Targeting Induced Local Lesions IN Genomes), which can be used to screen for mutations in a target region in pooled samples instead of in individual plants. TILLING has since been applied in investigations of a great number of animal and plant species (Taheri et al., 2017 2017 ). TILLING was initially developed for screening point mutations induced by chemical mutagenesis, mostly by ethyl methanesulfonate (EMS) (McCallum et al., 2000 2000 ; Till et al., 2003 2003 ). A TILLING system for the screening of large deletions induced by fast neutrons was developed and referred to as deletion TILLING (De-TILLING) (Rogers et al., 2009 2009 ). In addition, new TILLING variants, such as individualized TILLING (iTILLING) (Bush and Krysan, 2010 2010 ), TILLING by sequence (Seq-TILLING) (Tsai et al., 2011 2011 ; Kumar et al., 2017 2017 ), high-resolution melting (HRM)-TILLING (Dong et al., 2009 2009 ; Gady et al., 2009 2009 ), compressed sequencing (ComSeq)-TILLING (Nida et al., 2016 2016 ), and denaturing high-performance liquid chromatography (DHPLC)-based TILLING (Colasuonno et al., 2016 2016 ), were developed for simplicity or higher throughput levels. However, these systems have been applied only to EMS or fast neutron mutagenesis. HRM curve analysis, which is based on fluorescence changes during the melting of the DNA duplex, is a simple, cost-effective, and high-throughput method for mutation screening and genotyping (Ririe et al., 1997 1997 ). HRM has already been widely used in plant research for high-throughput genotyping, mapping genes, and testing food products and seeds (Simko, 2016 2016 ). It has also been used in combination with TILLING (HRM-TILLING) for screening EMS-mutagenized populations of tomato (Gady et al., 2009 2009 ), wheat (Dong et al., 2009 2009 ; Lochlainn et al., 2011 2011 ), and grapevine (Acanda et al., 2014 2014 ). As with most other TILLING systems, HRM-TILLING has been used only for the screening of EMS-induced mutations. γ irradiation has been used widely as an efficient tool for mutation induction in about 200 economically important plant species ( http://mvd.iaea.org http://mvd.iaea.org ). A great number of mutants have been generated for genetic studies and for the breeding of new cultivars that have been grown on billions of hectares (Ahloowalia et al., 2004 2004 ; Shu et al., 2012 2012 ). Both small insertion/deletions (Indels) and point mutations can be induced by γ ray (Nawaz and Shu, 2014 2014 ; Li et al., 2016 2016 ), which makes it a unique mutagen. As with other mutagens, γ radiation induces mutations only at low frequencies, such as 7.5×10 −6 in rice (Li et al., 2016 2016 ). Therefore, a TILLING system suitable for the high-throughput screening of both Indels and point mutations is needed to increase the selection efficiency of γ ray-induced mutations. Sato et al. ( 2006 2006 ) reported a simplified version of TILLING for the identification of γ ray-induced mutants in rice, in which crude extracts of Brassica rapa petioles were used as mismatch-specific endonucleases instead of celery endonuclease I (CELI), and DNA fragments were separated on agarose gels instead of using the expensive LI-COR system. However, its throughput capacity was still very low. Recently, Hwang et al. ( 2017 2017 ) identified a number of mutations in membrane transport genes in a γ ray-mutagenized rice population using TILLING. Thus, there is a need to establish new TILLING platforms suitable for more efficient and simpler screening of γ ray-induced mutations. In this study, we first investigated the power of HRM analysis for the detection of various rice ( Oryza sativa ) deletion mutations in pooled samples. Then, we applied the optimized system to screening for mutations in two genes in an M 2 rice population exposed to γ radiation. Materials and methods 1 Plant materials 2 Because γ rays can induce deletion mutations of different sizes, mutants with 1–11-bp deletions together with their wild-type (WT) parents were used for the HRM analysis of pooled samples (Table 1 1 , Fig. 1 1 ). The mutant lines included two low phytic acid lines (MH86- lpa and Z9B- lpa ), one lesion mimic mutant (JZBm), two bentazon susceptible mutants (GZ63m1 and GZ63m2), and one elongated uppermost internode mutant (Elong B). Exons and introns are to scale and are indicated by filled boxes and solid lines, respectively, for each gene, with deletion mutations represented by black triangles WT: wild-type; F: forward; R: reverse; T m : melting temperature Mutant WT parent Gene (LOC number) Deletion Primer (5'→3') T m (°C) Amplicon (WT) (bp) Reference 8 Mutants and WT parents 2 MH86- lpa 2 Minghui86 (MH86) 2 OsSultr3;3 (Os04g55800) 2 G F:CGACTTGAAGAAATCAACAGAGAAGCCTG 2 56.3 2 179 2 Zhao et al., 2008 2008 R:GGGGTTTACTATGCCACATAACT 2 JZBm 2 Jiazhe B (JZB) 2 CYP71A1 (Os12g16720) 2 G F:CTGGGACAACCCGCTGGAGT 2 62.0 2 288 2 Lu et al., 2016 2016 R:TTCCTGACGGCGACGAAGA 2 GZ63m1 2 Guangzhan 63 (GZ63) 2 CYP81A6 (Os03g55240) 2 CG F:GAAGCCGATGCACGCCACC 2 60.0 2 192 2 Wang et al., 2012 2012 R:GCGCCGTTGAACGAGACCAG 2 Elong B (ELB) 2 Kelong B (KLB) 2 CYP714D1 (Os05g40384) 2 TCT F:AAGCCCAAGTACCTCCAGAA 2 54.8 2 294 2 Fu et al., 2008 2008 R:CGAGTAATCACTCCCAAAGC 2 Z9B- lpa 2 Zhong 9B (Z9B) 2 OsSultr3;3 (Os04g55800) 2 AAATCC F:GGTGCCCAGCTACTCCTTCTC 2 59.0 2 98 2 Zhao et al., 2008 2008 R:GCGAAGATTATATCATTGCCTG 2 GZ63m2 2 Guangzhan 63 (GZ63) 2 CYP81A6 (Os03g55240) 2 ACCTTCGCGAA F:GAAGCCGATGCACGCCACC 2 60.0 2 192 2 Wang et al., 2012 2012 R:GCGCCGTTGAACGAGACCAG 8 8 M 2 population 2 DS552 M 2 2 DS552 2 OsLCT1 (Os06g38120) 2 F:CTCGATGTTAAGCATGCTCC 2 61.0 2 195 2 Uraguchi et al., 2011 2011 R:AGAGTCAGGAACGCGGCTAC 2 2 2 SPDT (Os06g05160) 2 F:TTCTCGGAGGAGGCTAAT 2 52.0 2 259 2 Yamaji et al., 2017 2017 R:CCACGCATTCTGGTTACAT The M 2 population of the japonica rice line DS552 was developed through γ ray ( 137 Cs) irradiation at the Irradiation Centre of the Zhejiang Academy of Agricultural Sciences (Hangzhou, China). A total dose of 100 Gy was applied for the treatment of dried seeds at a dose rate of about 1 Gy/min. Irradiated seeds were germinated and grown as an M 1 population at the experimental farm of Jiaxing Academy of Agricultural Sciences (Jiaxing, Zhejiang, China). M 2 seeds were bulk-harvested from the M 1 plants. For mutation screening, about 5000 M 2 seeds were grown into M 2 seedlings under hydroponic culture conditions with a culture solution modified from Yoshida et al. ( 1976 1976 ) in a glasshouse with a 12-h photoperiod (day (30±2) °C and night (24±2) °C). Seeds harvested from mutated M 2 plants were used to produce M 3 populations. For mutation screening, we selected two rice genes: rice low-affinity cation transporter 1 ( OsLCT1 , LOC_Os06g38120), which encodes a cadmium (Cd) transporter (Uraguchi et al., 2011 2011 ), and SULTR-like phosphorus (P) distribution transporter gene ( SPDT , LOC_Os06g05160), which controls the allocation of P to the rice grain (Yamaji et al., 2017 2017 ). DNA extraction 2 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 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 M2 seedlings was extracted using a simple, safe, and fast DNA extraction protocol adopted from Tan et al. ( 2016 2016 ). Briefly, leaf disks (diameter about 2 mm) were collected from samples of four M2 seedlings using a hole puncher, and then mixed and extracted into 96-well polymerase chain reaction (PCR) plates. HRM analysis 2 PCR primers for the amplification of fragments encompassing the mutations of six deletion materials (Table 1 1 ) were designed based on the genome sequence of the rice cultivar Nipponbare ( http://www.gramene.org http://www.gramene.org ). Based on the gene information on OsLCT1 and SPDT from the Gramene database (Fig. 1 1 ), primer pairs were designed in their respective exonic regions (Table 1 1 ). All of the primers were designed using Primer Premier 5 software and synthesized by Shanghai Sangong Biological Engineering Technology & Services Co., Ltd., China. To establish the HRM-TILLING, pooled DNA templates were produced by mixing WT and mutants at 1:1, 3:1, 7:1, and 15:1 ratios. For the mutation screening, pooled DNA extracted directly from each batch of four M 2 plants was used. PCRs were performed in a 10-µl volume with 25 ng of mixed DNA, 5 µl of 2× Master Mix (containing 2× PCR buffer, 4 mmol/L MgCl 2 , 0.4 mmol/L 2'-deoxyribonucleoside triphosphates (dNTPs), and 50 U/ml Taq DNA polymerase; Toyobo Co., Ltd., Japan), 0.2 µl each of 10 μ mol/L primers, and 1 µl of 10× EvaGreen (Biotium, USA), covered with a drop of mineral oil to prevent solution evaporation. The WT and mutants were used in each run as controls. The following PCR conditions were used: 5 min at 94 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at 52–62 °C, and 30 s at 72 °C, with a final extension at 72 °C for 8 min and a hold at 16 °C. The annealing temperature was optimized for each particular gene and fragment (Table 1 1 ). Following PCR, plates were transferred to a LightScanner (Idaho Technology Inc., USA) and subjected to HRM analysis according to Tan et al. ( 2016 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.). The WTs were used as references, and samples with relative fluorescence differences (∆ F ) of > 0.05 were considered to be significantly different from the reference (Hofinger et al., 2009 2009 ). Validation by Sanger sequencing 2 For mixed samples that had ∆ F values > 0.05 when compared with the WT parent “DS552”, the genomic DNA of each of the four plants was individually extracted using the CTAB method. The target fragments were amplified by PCR in a 50-µl volume, separated on 1.0% (0.01 g/ml) agarose gels, recovered using a DNA Gel Extraction Kit (Axygen Biotechnology Co., Ltd., Hangzhou, China), and subsequently sequenced by Sanger sequencing by Tsingke Biological Technology Co., Ltd. (Hangzhou, China). Because the sequencing results showed mixed peaks, clone sequencing was performed. Results 1 Establishment of an HRM-TILLING system for screening deletion mutations 2 To establish an HRM-TILLING system suitable for screening Indel mutants in pooled DNA samples, the identification of a proper pooling ratio of Indel mutants to WT samples was key. Thus, the DNA of each of six Indel mutants was mixed with WT DNA in different ratios and subjected to HRM analyses. All six mutants, except the mutant of Zhong 9B (Z9B- lpa ), had HRM curves significantly different from that of the WT, with peak fluorescence differences (∆ F values) equal to or greater than 0.05. Z9B- lpa had an HRM curve most similar to its WT, with the ∆ F being only about 0.03 (Fig. 2 2 ). WT samples were used as references for each analysis and are shown as horizontal lines. Fluorescence difference curves are automatically grouped by the HRM system and are indicated by different colors. P1:1, 3:1, 7:1, and 15:1 stand for the mixture ratios of WT:MT HRM curves of the pooled samples were all significantly different from those of the WTs, with ∆ F values equal to or greater than 0.05 when the ratio was less than 7:1. In most cases, the differences between the pooled and the WT samples were greater than those between the mutants and the WT lines (Fig. 2 2 ). There were two types of differences between pooled and WT samples. The first type consisted of mutant line samples that showed decreasing ∆ F values from WT as their proportion in the pool decreased, including MH86- lpa , JZBm, ELB, and GZ63m2. These samples had ∆ F values close to 0.05 at the 7:1 (WT: mutant) ratio. The second type included mutant lines GZ63m1 and Z9B- lpa . Their pooled samples showed increasing ∆ F values when compared with the WT, as their proportion in the pool decreased (Fig. 2 2 ). The sharp difference between the two types of samples appeared to be mutation/amplicon specific, because samples within each type had different deletion sizes and amplicon lengths (Table 1 1 ). Furthermore, except for GZ63m2, the ∆ F values resulting from the deletions were mostly revealed at high temperatures of 90.0–94.0 °C. In addition, all but one mutant, Z9B- lpa , had positive ∆ F values, suggesting that these deletion mutations resulted in more fluorescence dye integrating into the amplicons. These results demonstrated that pooled samples with 1/8 mutant DNA could be differentiated from WT DNA by HRM analysis. Because M 2 plants could be heterozygous for induced mutations, pooling of four M 2 plants was suitable for HRM-based mutation screening. HRM-TILLING of OsLCT1 and SPDT 2 From about 5000 M 2 seeds, 4560 seedlings were established and grown hydroponically. A total of 1140 pooled DNA samples were produced and subjected to HRM analysis. Most samples had melting curves not significantly different from “DS552” (∆ F < 0.05); however, three samples, two for OsLCT1 and one for SPDT , showed significantly different HRM curves with ∆ F values of > 0.05 at temperatures of 90.0–92.0 °C and 81.5–83.5 °C, respectively (Fig. 3 3 ). The wild-type (WT, horizontal line) was chosen as the reference for the development of fluorescence difference curves. The mutants (red lines) were automatically sorted by the system because their HRM curves had ∆ F values of > 0.05 from the WT curves The 12 seedlings represented in these three pooled samples were sequenced for the respective fragments. Three seedlings had sequencing chromatograms that contained either a single double peak (Fig. 4a 4a ) or double peaks after a particular nucleotide (Figs. 4b and 4c 4b and 4c ), suggesting that they carried mutations in the heterozygous state. Thus, the amplicons of these seedlings were subjected to clone sequencing. Finally, one G→A single nucleotide substitution at the 4304 bp and one single nucleotide A insertion at the 4240 bp of OsLCT1 were identified. Also, one TTC trinucleotide deletion was identified at the position of 5948–5950 bp of SPDT . Based on their positions in their respective genes, the G→A mutation is a synonymous mutation that does not change the amino acid; the single A insertion causes a frame shift, resulting in a truncated protein due to an early stop codon at 4399 bp; and the TTC deletion of SPDT occurs in the 3' untranslated region (3' UTR). (a, b) OsLCT1 ; (c) SPDT . The rectangular box in (a) indicates a heterozygous site with a mutation of G→A; a single A insertion in (b) is indicated by an arrow, and a TTC deletion in (c) is indicated by a black line Isolation of homozygous M 3 mutant plants 2 An M 3 population was developed from seeds harvested from an M 2 plant carrying a heterozygous A insertion mutation and was subjected to HRM analysis, together with the WT parent “DS552”. Plants were divided into three groups based on their |Δ F | values compared with the WT. Group I plants were indistinguishable from “DS552”, suggesting that they were homozygous WT lines; Group II plants were similar to the M 2 plant, suggesting that they were heterozygous mutant lines; and Group III plants differed from Groups I and II, suggesting that they were homozygous mutant lines (Fig. 5 5 ). The wild-type parent “DS552” was used as the reference for fluorescence curve development and is shown as the horizontal line (Group I). The curve of the M 2 plant is indicated in black (Group II), curves of heterozygous M 3 plants in red, and curves of homozygous M 3 plants in blue (Group III) Discussion 1 The invention of TILLING has significantly advanced functional genomics by efficiently incorporating classical mutagenesis into a modern gene function analysis. However, its application in γ ray mutagenesis has, to date, been limited. In this study we have established an HRM-based TILLING system and demonstrated its suitability for screening mutations in γ ray-mutagenized M 2 plants. Detection of Indel mutations in pooled samples 2 The detection of mutations in pooled samples is at the core of TILLING systems. In mismatch cleavage-based systems (using CELI or other endonucleases), the mutation detection limit is about 1 in 16. Thus, pooling eight M 2 plants is recommended because mutations often exist in the heterozygous state (Colbert et al., 2001 2001 ). In De-TILLING (Rogers et al., 2009 2009 ) and Seq-TILLING (Tsai et al., 2011 2011 ), more M 2 plants can be pooled because these techniques have a greater capability for identifying mutations in pooled samples. Although HRM analysis has the advantage of being high throughput, its capability for identifying single nucleotide polymorphisms (SNPs) in pooled samples is not as good as that of CELI-based mismatch detection. Thus, the pooling of samples of four M 2 plants was recommended for detecting mutations in EMS-mutagenized populations (Lochlainn et al., 2011 2011 ). In the present study, we first demonstrated that Indel mutations could be efficiently identified in pooled samples when the WT:mutant ratio was higher than 7:1. Unexpectedly, the detection power was much higher for certain mutations. For example, GZ63m1 and Z9B- lpa had HRM curves that were even more different from WT when the WT:mutant ratio was lower (Fig. 2 2 ). This appears to be unique to Indel mutations because this phenomenon was never reported in HRM-TILLING studies characterizing single base-pair substitutions (Gady et al., 2009 2009 ; Botticella et al., 2011 2011 ; Lochlainn et al., 2011 2011 ; Acanda et al., 2014 2014 ; Bovina et al., 2014 2014 ). However, in HRM analysis of nine Indel mutations using mixed samples, Cousins et al. ( 2013 2013 ) also observed one mutation (1-bp deletion) that performed similarly to GZ63m1 and Z9B- lpa , i.e. the HRM difference between the mixture samples and the WT increased as the WT:mutant ratio decreased from 1:1 to 1:9. While its underlying mechanism is still unknown, this characteristic makes HRM-TILLING more powerful for detecting Indel mutations than for detecting point mutations in pooled samples. A number of factors can affect the HRM curves, including DNA amplicon size and sequence context, and GC content and distribution (Mader et al., 2008 2008 ). In the present study, the amplicons had lengths from 98 bp (Z9B- lpa ) to 294 bp (ELB) and GC content from 39.66% (MH86- lpa ) to 70.14% (JZBm), representing the most targets that may be searched using an HRM-TILLING analysis (Reed et al., 2007 2007 ). This was also reflected by the different shapes and differentiating temperature regimes of fluorescence in the present study (Fig. 2 2 ). Because HRM analysis is not suitable for long amplicons, the target fragment size of HRM-TILLING is often less than 400 bp (Wittwer et al., 2003 2003 ), which is only about 1/4 to 1/3 the length of target fragments analyzed by classical CELI-based TILLING (Till et al., 2003 2003 ). However, DNA extraction, PCR, and melting curve analyses can all be performed in 96-well plates in HRM-TILLING, which enables very high-throughput mutation screening. For example, it took only 18 d to complete the analysis of the two genes in the 4560 M 2 seedlings in the present study. Type, frequency, and potential uses of identified mutations 2 From the 4560 M 2 seedlings, two Indel mutations, which were both heterozygous, were identified by HRM-TILLING. Based on the length of the amplicons, 195 bp for OsLCT1 and 259 bp for SPDT , and the size of rice genome, about 373 Mbp, this amounts to a mutation rate of 0.97×10 –6 per nucleotide. This mutation rate is similar to those reported for Indels in γ ray-induced M 2 plants (1.1×10 −6 –1.3×10 −6 ) (Li et al., 2016 2016 ). Only one nucleotide substitution was identified in the present study; therefore, there was a substitution mutation rate of 0.33×10 −6 per nucleotide. The substitution mutation rate was much lower than those estimated by whole genome sequencing (4.7×10 −6 –7.9×10 −6 ) (Li et al., 2016 2016 ). Also, it is possible that a small proportion of substitution mutations were missed because certain mutations are difficult to identify using a routine HRM analysis (Zhang et al., 2014 2014 ). However, this does not suggest that our HRM-TILLING method missed a large proportion of base substitution mutations. Because mutations do not occur evenly in all genomic regions, mutation frequencies, when estimated on the basis of individual genes or genomic fragments, could vary greatly for different genes in the same mutated population. For example, Hwang et al. ( 2017 2017 ) observed mutation frequencies from 0 to 3.38×10 −6 for different genes (1.52×10 −6 on average) in a γ ray-irradiated rice mutant population. Variation in mutation frequency among different genes was also reported in EMS-mutagenized rice populations, such as frequencies of 0.87×10 −6 –6.08×10 −6 per nucleotide for the eight genes screened by Till et al. ( 2007 2007 ). Among the 41 mutations identified by Hwang et al. ( 2017 2017 ), only 1 was a deletion mutation (1 bp). The low proportion of this type of mutation and its single base-pair deletion suggest that CELI-based TILLING might be more suited to identifying base substitution mutations. However, more studies are needed to ascertain whether CELI-based TILLING could miss deletion mutations. In the present study, OsLCT1 and SPDT were chosen to demonstrate the effectiveness of our technique because mutations of both genes are expected to generate “promising alleles” for breeding healthy rice grains. OsLCT1 is crucial for Cd transport into rice grains and, thus, a mutation of OsLCT1 could block transport and generate “low-Cd rice” (Uraguchi et al., 2011 2011 ). SPDT is responsible for channeling P from nodes to rice grains, and knockout or knockdown mutations of SPDT reduces the total P and phytic acid content of rice grains (Yamaji et al., 2017 2017 ). The 1-bp insertion mutation truncates OsLCT1 and thus would be valuable for breeding low-Cd rice. 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Gene identification and allele-specific marker development for two allelic low phytic acid mutations in rice (Oryza sativa L.) Compliance with ethics guidelines Shan LI, Song-mei LIU, Hao-wei FU, Jian-zhong HUANG, and Qing-yao SHU declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1700414 Project supported by the National Key Research and Development Program of China (No. 2016YFD0102103) 2017-08-19 2017-10-22 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2018 2018 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 19