Abstract
Discovering new genetic mutations is vital for expanding genetic resources for exploring the functions and breeding applications of genes. In this study, we created new mutant populations of rice and evaluated its effectiveness by using a chemical mutagen diepoxybutane (DEB) with the expectation of manageably causing small-scale deletions that induce knock-out mutations. Compared with the more common mutagen, ethyl methanesulfonate (EMS), DEB exhibited approximately 160 times the adverse impact on the early growth of rice. At 0.3 mM, which was 1/160 the concentration of EMS in the control population, the heading date in the DEB-mutated population showed dispersion, albeit with a small standard deviation. Therefore, similar to EMS, DEB has been shown to induce DNA mutations. According to the screening of waxy mutants using the Targeting Induced Local Lesions in Genomes (TILLING) method, DEB-mutated populations had nearly similar mutation frequencies to those of EMS-mutated populations. Therefore, we successfully isolated five mutant lines from the DEB-mutated population. Some of these mutants exhibited a low-amylose phenotype, which is applicable to breeding leading to enhanced taste evaluation. To utilize these mutated alleles, we developed co-dominant DNA markers. In this study, EMS induced transition mutations, as previously reported. In contrast, DEB specifically induced transversion mutations rather than small-scale deletions contrary to our initial expectations. These results demonstrate that DEB has a novel point of action for mutation and is useful for expanding genetic resources for crops.
This is a preview of subscription content, log in via an institution to check access.
References
Anai T (2012) Potential of a mutant-based reverse genetic approach for functional genomics and molecular breeding in soybean. Plant Breed 61:462–467. https://doi.org/10.1270/jsbbs.61.462
Anai T (2015) Mutant-based reverse genetics for functional genomics of non-model crops. In: Al-Khayri JM, Jaon SM, Johnson DV (eds) Advances in plant breeding strategies: breeding biotechnology and molecular tools. Springer, Switzerland, pp 473–487
Barstead RJ, Moerman DG (2006) C. elegans deletion mutant screening. In: Strange K (ed) C. elegans. Method and applications, methods in molecular biology, vol 351. Humana Press, USA, pp 51–58
Bolger ME, Weisshaar B, Scholz U, Stein N, Usadel B, Mayer KF (2014) Plant genome sequencing—application for crop improvement. Curr Opin Biotechnol 26:31–37. https://doi.org/10.1016/j.copbio.2013.08.019
Chaudhary J, Deshmukh R, Sonah H (2019) Mutagenesis approaches and their role in crop improvement. Plants 8:467. https://doi.org/10.3390/plants8110467
Fujita N, Kubo A, Francisco PB Jr, Nakakita M, Harada K, Minaka N, Nakamura Y (1999) Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice. Planta 208:283–293. https://doi.org/10.1007/s004250050560
Fujita N, Yoshida M, Asakura N, Ohdan T, Miyao A, Hirochika H, Nakamura Y (2006) Functional and characterization of starch synthase I using mutants in rice. Plant Physiol 140:1070–1084. https://doi.org/10.1104/pp.105.071845
Goto H, Asanome N, Suzuki K, Sano T, Saito H, Abe Y, ChubaNishio MT (2014) Objective evaluation of whiteness of cooked rice and rice cakes using a portable spectrophotometer. Breed Sci 63:489–494. https://doi.org/10.1270/jsbbs.63.489
Greene EA, Codomo CA, Taylor NE, Henikoff JG, Till BJ, Reynolds SH, Enns LC, Burtner C, Johnson JE, Odden AR, Comai L, Henikoff S (2003) Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164:731–740
Hoshino T, Iijima N, Hata M, Watanabe A, Kawakami T, Anai T (2020) Molecular characterization of high stearic acid soybean mutants and post-transcriptional control of GmSACPD genes in the mutant with a single nucleotide deletion. Plant Gene 21:100207. https://doi.org/10.1016/j.plgene.2019.100207
Hoshino T, Takagi Y, Anai T (2010) Novel GmFAD2-1b mutant alleles created by reverse genetics induce marked elevation of oleic acid content in soybean seeds in combination with GmFAD2-1a mutant alleles. Plant Breed 60:419–425. https://doi.org/10.1270/jsbbs.60.419
Hoshino T, Watanabe S, Takagi Y, Anai T (2014) A novel GmFAD3-2a mutant allele developed through TILLING reduces α-linolenic acid content in soybean seed oil. Plant Breed 64:371–377. https://doi.org/10.1270/jsbbs.64.371
Kyriakidou M, Tai HH, Anglin NL, Ellis D, Strömvik MV (2018) Current strategies of polyploid plant genome sequence assembly. Front Plant Sci 9:1660. https://doi.org/10.3389/fpls.2018.01660
Kumawat S, Rana N, Bansal R, Vishwakarma G, Mehetre ST, Das BK, Kumar M, Yadav SK, Sonah H, Sharma TR, Deshmukh R (2019) Expanding avenue of fast neutron mediated mutagenesis for crop improvement. Plants 8:164. https://doi.org/10.3390/plants8060164
Leung H, Wu C, Baraoidan M, Bordeos A, Ramos M, Madamba S, Cabauatan P, Vera Cruz C, Portugal A, Reves G, Bruskiewich R, McLaren G, Gregoria G, Bennett J, Brar D, Khush G, Schnable P, Wang G, Leach J (2001) Deletion mutants for functional genomics: progress in phenotyping, sequence assignment, and database development. In: Khush GH, Brar DS, Hardy B (eds) Rice genetics IV, rice genetics collection. World Scientific, Singapore, pp 239–251
Li S, Zhang N, Li J, Xiang J (2019) A new male sterile line of “Duanye-13” radish (Raphanus sativus L.) produced by ethyl methanesulfonate mutagenesis. Genet Resour Crop Evol 66:981–987. https://doi.org/10.1007/s10722-019-00772-y
Li S, Zhong X, Zhang X, Rahman MM, Lan J, Tang H, Qi P, Ma J, Wang J, Chen G, Lan X, Deng M, Li Z, Harwood W, Lu Z, Wei Y, Zheng Y, Jiang Q (2020) Production of waxy tetraploid wheat (Triticum turgidum durum L.) by EMS mutagenesis. Genet Resour Crop Evol 67:433–443. https://doi.org/10.1007/s10722-019-00875-6123458697(),-volV
Lin B, Hsu Y, Kuo S, Lin Y, Wu Y, Kuo C (2017) Novel Wx alleles induced by chemical mutagens in rice, Oryza sativa L. Plant Breed 136:206–213. https://doi.org/10.1111/pbr.12449
Mba C, Afza R, Bado S, Jain SM (2010) Induced mutagenesis in plant using physical and chemical agents. In: Devey MR, Anthony P (eds) Plant cell culture: essential methods. Wiley, USA, pp 77–89
Mikami I, Uwatoko N, Ikeda Y, Yamaguchi J, Hirano HY, Suzuki Y, Sano Y (2008) Allelic diversification at the wx locus in landraces of Asian rice. Theor Appl Genet 116:979–989. https://doi.org/10.1007/s00122-008-0729-z
Miyao A, Tanaka K, Murata K, Sawaki H, Takeda S, Abe K, Shinozaka Y, Onosato K, Hirochika H (2003) Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome. Plant Cell 15:1771–1780. https://doi.org/10.1105/tpc.012559
Morita R, Kusaba M, Iida S, Yamaguchi H, Nishio T, Nishimura M (2009) Molecular characterization of mutations induced by gamma irradiation in rice. Genes Genet Syst 84:361–370. https://doi.org/10.1266/ggs.84.361
Neff MM, Turk E, Kalishman M (2002) Web-based primer design for single nucleotide polymorphism analysis. Trends Genet 18:613–615. https://doi.org/10.1016/S0168-9525(02)02820-2
Nishizawa-Yokoi A, Cermak T, Hoshino T, Sugimoto K, Saika H, Mori A, Osakabe K, Hamada M, Katayose Y, Starker C, Voytas DF, Toki S (2016) A defect in DNA ligase4 enhances the frequency of TALEN-mediated targeted mutagenesis in rice. Plant Physiol 170:653–666. https://doi.org/10.1104/pp.15.01542
Pérez L, Soto E, Farré G, Juanos J, Villorbina G, Bassie L, Medina V, Serrato AJ, Sahrawy M, Rojas JA, Romagosa I, Muñoz P, Zhu C, Christou P (2019) CRISPR/Cas mutations in the rice Waxy/GBSSI gene induce allele-specific and zygosity-dependent feedback effects on endosperm starch biosynthesis. Plant Cell Rep 38:417–433. https://doi.org/10.1007/s00299-019-02388-z
Ram H, SalviSoni PP, Gandass N, Sharma A, Kaur A, Sharma TR (2019) Insertional mutagenesis approaches and their use in rice for functional genomics. Plants 8:310. https://doi.org/10.3390/plants8090310
Satoh H, Matsusaka H, Kumamaru T (2010) Use of N-methyl-N-nitrosourea treatment of fertilized egg cells for saturation mutagenesis of rice. Breed Sci 60:475–485. https://doi.org/10.1270/jsbbs.60.475
Serrat X, Esteban R, Guibourt N, Moysset L, Nogués S, Lalanne E (2014) EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations. Plant Methods 10:5. https://doi.org/10.1186/1746-4811-10-5
Shibutani S, Takeshita M, Grollman AP (1991) Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature 349:431–434. https://doi.org/10.1038/349431a0
Sprink T, Metje J, Hartung F (2015) Plant genome editing by novel tools: TALEN and other sequence specific nuclease. Curr Opin Biotechnol 32:47–53. https://doi.org/10.1038/nprot.2006.329
Suzuki T, Eiguchi M, Kumamaru T, Satoh H, Matsusaka H, Moriguchi K, Nagato Y, Kurata N (2008) MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice. Mol Genet Genomics 279:213–223. https://doi.org/10.1007/s00438-007-0293-2
Tajiri T, Maki H, Sekiguchi M (1995) Functional cooperation of MutT, MutM and MutY proteins in preventing mutations caused by spontaneous oxidation of guanine nucleotide in Escherichia coli. Mutant Res 336:257–267. https://doi.org/10.1016/0921-8777(94)00062-B
Tian Z, Qian Q, Liu Q, Yan M, Liu X, Yan C, Liu G, Gao Z, Tang S, Zeng D, Wang Y, Yu J, Gu M, Li J (2009) Allelic diversities in rice starch biosynthesis lead to a diverse array of rice eating and cooking qualities. Proc Natl Acad Sci USA 106:21760–21765. https://doi.org/10.1073/pnas.0912396106
Till BJ, Cooper J, Tai TH, Colowit P, Greene EA, Henikoff S, Comai L (2007) Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol 11:7–19. https://doi.org/10.1186/1471-2229-7-19
Till BJ, Zerr T, Comai L, Henikoff S (2006) A protocol for TILLING and Ecotilling in plants and animals. Nat Protoc 1:2465–2477. https://doi.org/10.1038/nprot.2006.329
Wang ZY, Wu ZL, Xing YY, Zheng FG, Guo XL, Zhang WG, Hong MM (1990) Nucleotide sequence of rice waxy gene. Nucleic Acid Res 18:5898. https://doi.org/10.1093/nar/18.19.5898
Woo JW, Kim J, Kwon SI, Corvalán C, Cho SW, Kim H, Kim SG, Kim ST, Choe S, Kim JS (2015) DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol 33:1162–1164. https://doi.org/10.1038/nbt.3389
Wu JL, Wu C, Lei C, Baraoidan M, Bordeos A, Madamba MR, Ramos-Pamplona M, Mauleon R, Portugal A, Ulat VJ, Bruskiewich R, Wang G, Leach J, Khush G, Leung H (2005) Chemical- and irradiation-induced mutants of indica rice IR64 for forward and reverse genetics. Plant Mol Biol 59:85–97. https://doi.org/10.1007/s11103-004-5112-0
Zhang C, Zhu J, Chen S, Fan X, Li Q, Lu Y, Wang M, Yu H, Yi C, Tang S, Gu M, Liu Q (2019) Wxlv, the ancestral allele of rice Waxy gene. Mol Plant 12:1157–1166. https://doi.org/10.1016/j.molp.2019.05.011
Acknowledgements
We would like to express our thanks to Professor Naoko Fujita of the Akita Prefecture University for providing antibody against Waxy protein of rice and kind advice on western blotting. We are grateful to Kazuma Abe, Eriko Ishikawa, Kana Takiguchi, Anri Watanabe, Takuya Yoshida, Chiho Kamimura, Asami Hashimoto, Nobushige Iijima, and Masakazu Hata of the laboratory of Crop Breeding, Faculty of Agriculture, Yamagata University for their technical assistance. We also thank Hideharu Homma, Yoichi Kikuchi, Kenichi Tanaka, Takuya Sakuma, Kazuhiro Ariga, and Ayaka Yamazaki of the Field Science Center, Faculty of Agriculture, Yamagata University for their support in field management.
Funding
This work was supported partially by JSPS KAKENHI Grant Nos. 25892005 and 17K08164 to TH, and YU-COE(M) Project from Yamagata University to MW. This work also supported by the Urakami Foundation for Food and Food Culture Promotion and Tobe Maki Scholarship Foundation to TH.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
10722_2020_950_MOESM1_ESM.eps
Fig. S1 Allelic mutations in waxy gene identified from the EMS M2 (n = 7,354) and DEB M2 (n = 2,777) populations through the TILLING method. The schematic gene structure of waxy gene is shown. Boxes represent exons (white boxes, untranslated regions; blue (EMS) and red (DEB), open reading frames), and lines between boxes represent introns. Nucleotide substitutions between the mutants and original cultivar Tsuyahime are indicated. The numbers of a mutated nucleotide of the mutation in exons represent the distance from the first nucleotide of the initial codon. Amino acid substitutions between the mutants and original cultivar Tsuyahime are also indicated (EPS 2322 kb)
10722_2020_950_MOESM2_ESM.eps
Fig. S2 Expression analysis of the waxy gene and quantitative determination of the Waxy protein in developing endosperm of the original cultivar Tsuyahime and the synonymous mutant 14-1478 line. (A) Left, 2 weeks after heading; Right, 4 weeks after heading). Quantitative RT–PCR analysis of the Waxy gene in developing endosperms of the original cultivar Tsuyahime (Red) and 14-1478 line (White). Expression levels are presented as ratios relative to OsUBQ2 gene expression. *Student’s t-test, *P < 0.05. (B) SDS-PAGE (bottom, stained with Coomassie Brilliant Blue) of Soluble proteins (SP) and Western blot (top, detected with anti-Waxy protein) of the Tight Binding Protein (TBP) from developing endosperm of the original cultivar Tsuyahime and the synonymous mutant 14-1478 line are shown. The amount of total SP was used as a loading control. The picture shows a repetition of two different sample sets (EPS 1491 kb)
Rights and permissions
About this article
Cite this article
Kawakami, T., Goto, H., Abe, Y. et al. High frequency of transversion mutations in the rice (Oryza sativa L.) mutant population produced by diepoxybutane mutagenesis. Genet Resour Crop Evol 67, 1355–1365 (2020). https://doi.org/10.1007/s10722-020-00950-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10722-020-00950-3