Abstract
Fast neutron (FN) bombardment is a powerful mutagen that can be effectively employed for functional genomics studies in the post-genome era. In soybean, dwarfism is a desirable agricultural characteristic that improves lodging resistance. In the present study, we selected a dwarf mutant soybean among approximately 10,000 M4 progeny lines derived from FN-irradiated seeds of cultivar Williams 82. This dwarf mutant exhibited reduced plant height, only approximately 20 % of wild type. Using mutant plants homozygous for this dwarf phenotype, we performed whole genome sequencing by Illumina HiSeq to identify the deletion site responsible for the dwarfism. Comparative sequence analysis by mapping the mutant reads to the soybean reference genome sequence (wild type) predicted 13 large deletion regions. Among these, three loci (designated del1–3, del2–3, and del3–15) were validated by two complementary PCRs using two allele-specific reverse primers, respectively. We found that the del1–3 and del2–3 loci are positioned in non-coding regions of chromosome 3. In del3–15, the mutated allele has an 803-bp deletion including the first partial exon of Glyma15g05831 (peroxidase superfamily protein) on chromosome 15, resulting in the loss of a start codon. Reverse transcription-PCR analysis revealed that the expression of the gene Glyma15g05831 was completely abolished in the dwarf mutant. A lack of peroxidase (which catalyzes the generation of reactive oxygen species) coupled with indole-3-acetic acid oxidation may be responsible for the dwarfing of this mutant.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Belfield EJ, Gan X, Mithani A, Brown C, Jiang C, Franklin K, Alvey E, Wibowo A, Jung M, Bailey K, Kalwani S, Ragoussis J, Mott R, Harberd NP (2012) Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana. Genome Res 22:1306–1315. doi:10.1101/gr.131474.111
Bolon Y-T, Haun WJ, Xu WW, Grant D, Stacey MG, Nelson RT, Gerhardt DJ, Jeddeloh JA, Stacey G, Muehlbauer GJ, Orf JH, Naeve SL, Stuper RM, Vance CP (2011) Phenotypic and genomic analyses of a fast neutron mutant population resource in soybean. Plant Physiol 156:240–253. doi:10.1104/pp.110.170811
Bruce M, Hess A, Bai J, Mauleon R, Diaz MG, Sugiyama N, Bordeos A, Wang G-L, Leung H, Leach JE (2009) Detection of genomic deletions in rice using oligonucleotide microarrays. BMC Genomics 10:129. doi:10.1186/1471-2164-10-129
Bruggemann E, Handwerger K, Essex C, Storz G (1996) Analysis of fast neutron-generated mutants at the Arabidopsis thaliana HY4 locus. Plant J 10:755–760
Byth DE, Weber CR (1969) Two mutant genes causing dwarfness in soybeans. J Hered 60:278–280
Cooper RL, Martin RJ, Schmitthenner AF, McBlain BA, Fioritto RJ, St. Martin SK, Calip-DuBois A (1991) Registration of ‘Hobbit 87’ soybean. Crop Sci 31:1093
Cooper RL, Martin RJ, St. Martin SK, Calip-DuBois A, Fioritto RJ, Schmitthenner AF (1995) Registration of ‘Charleston’ soybean. Crop Sci 35:593
Cooper RL, Mendiola T, St. Martin SK, Fioritto RJ, Schmitthenner AF, Dorrance AE (2001) Registration of ‘Strong’ soybean. Crop Sci 41:921–922
Cooper RL, Mendiola T, St. Martin SK, Fioritto RJ, Dorrance AE (2003) Registration of ‘Apex’ soybean. Crop Sci 43:1563
Fehr WR (1972) Inheritance of a mutation for dwarfness in soybeans. Crop Sci 2:212–213
Hedden P (2003) The genes of the green revolution. Trends Genet 19:5–9
Hiraga S, Sasaki K, Ito H, Ito H, Ohashi Y, Matsui H (2001) A large family of class III plant peroxidases. Plant Cell Physiol 42:462–468
Hoffmann D, Jiang Q, Men A, Kinkema M, Gresshoff PM (2007) Nodulation deficiency caused by fast neutron mutagenesis of the model legume Lotus japonicus. J Plant Physiol 164:460–469. doi:10.1016/j.jplph.2006.12.005
Kawano T (2003) Roles of the reactive oxygen species-generating peroxidase reactions in plant defense and growth induction. Plant Cell Rep 21:829–837. doi:10.1007/s00299-003-0591-z
Kuromori T, Takahashi S, Kondou Y, Shinozaki K, Matsui M (2009) Phenome analysis in plant species using loss-of-function and gain-of-function mutants. Plant Cell Physiol 50:1215–1231. doi:10.1093/pcp/pcp078
Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows–Wheeler Transform. Bioinformatics 25:1754–1760
Li X, Zhang Y (2002) Reverse genetics by fast neutron mutagenesis in higher plants. Funct Integr Genomics 2:254–258. doi:10.1007/s10142-002-0076-0
Li X, Song Y, Century K, Straight S, Ronald P, Dong X, Lassner M, Zhang Y (2001) A fast neutron deletion mutagenesis-based reverse genetics system for plants. Plant J 27:235–242
Li X, Lassner M, Zhang Y (2002) Deleteagene: a fast neutron deletion mutagenesis-based gene knockout system for plants. Comp Funct Genomics 3:158–160. doi:10.1002/cfg.148
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J et al (2009) 1000 Genome project data processing subgroup. The sequence alignment/map (SAM) format and SAM tools. Bioinformatics 25:2078–2079
Miller G, Suzuki N, Rizhsky L, Hegie A, Koussevitzky S, Mittler R (2007) Double mutants deficient in cytosolic and thylakoid ascorbate peroxidase reveal a complex mode of interaction between reactive oxygen species, plant development, and response to abiotic stresses. Plant Physiol 144:1777–1785. doi:10.1104/pp.107.101436
Morozova O, Marra MA (2008) Applications of next-generation sequencing technologies in functional genomics. Genomics 92:255–264. doi:10.1016/j.ygeno.2008.07.001
Multani DS, Briggs SP, Chamberlin MA, Blakeslee JJ, Murphy AS, Johal GS (2003) Loss of an MDR transporter in compact stalks of maize br2 and sorghum dw3 mutants. Science 302:81–84. doi:10.1126/science.1086072
Oldroyd GED, Long SR (2003) Identification and characterization of nodulation-signaling pathway 2, a gene of Medicago truncatula involved in Nod factor signaling. Plant Physiol 131:1027–1032. doi:10.1104/pp.102.010710
Peng J, Richards DE, Hartley NM, Murphy GP, Devos KM, Flintham JE, Beales J, Fish LJ, Worland AJ, Pelica F, Sudhakar D, Christou P, Snape JW, Gale MD, Harberd NP (1999) “Green revolution” genes encode mutant gibberellin response modulators. Nature 400:256–261. doi:10.1038/22307
Pnueli L, Liang H, Rozenberg M, Mittler R (2003) Growth suppression, altered stomatal responses, and augmented induction of heat shock proteins in cytosolic ascorbate peroxidase (Apx1)-deficient Arabidopsis plants. Plant J 34:185–201
Prendergast JG, Campbell H, Gilbert N, Dunlop MG, Bickmore WA, Semple CA (2007) Chromatin structure and evolution in the human genome. BMC Evol Biol 7:72. doi:10.1186/1471-2148-7-72
Rogers C, Wen J, Chen R, Oldroyd G (2009) Deletion-based reverse genetics in Medicago truncatula. Plant Physiol 151:1077–1086. doi:10.1104/pp.109.142919
Rosa SB, Caverzan A, Teixeira FK, Lazzarotto F, Silveira JAG, Ferreira-Silva SL, Abreu-Neto J, Margis R, Margis-Pinheiro M (2010) Cytosolic APx knockdown indicates an ambiguous redox responses in rice. Phytochemistry 71:548–558. doi:10.1016/j.phytochem.2010.01.003
Rozen S, Skaletsky H (1999) Primer3 on the WWW for general users and for biologist programmers. In: Misener S, Krawetz S (eds) Bioinformatics methods and protocols SE-20. Humana Press, Totowa, pp 365–386
Salvi S, Tuberosa R (2005) To clone or not to clone plant QTLs: present and future challenges. Trends Plant Sci 10:297–304. doi:10.1016/j.tplants.2005.04.008
Sasaki A, Ashikari M, Ueguchi-Tanaka M, Itoh H, Nishimura A, Swapan D, Ishiyama K, Saito T, Kobayashi M, Khush GS, Kitano H, Matsuoka M (2002) A mutant gibberellin-synthesis gene in rice. Nature 416:701–702. doi:10.1038/416701a
Savitsky PA, Gazaryan IG, Tishkov VI, Lagrimini LM, Ruzgas T, Gorton L (1999) Oxidation of indole-3-acetic acid by dioxygen catalysed by plant peroxidases: specificity for the enzyme structure. Biochem J 340:579–583
Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, Hyten DL, Song Q, Thelen JJ, Cheng J, Xu D, Hellsten U, May GD, Yu Y, Sakurai T, Umezawa T, Bhattacharyya MK, Sandhu D, Valliyodan B, Lindquist E, Peto M, Grant D, Shu S, Goodstein D, Barry K, Futrell-Griggs M, Abernathy B, Du J, Tian Z, Zhu L, Gill N, Joshi T, Libault M, Sethuraman A, Zhang X-C, Shinozaki K, Nguyen HT, Wing RA, Cregan P, Specht J, Grimwood J, Rokhsar D, Stacey G, Shoemaker RC, Jackson SA (2010) Genome sequence of the palaeopolyploid soybean. Nature 463:178–183. doi:10.1038/nature08670
Searle IR, Men AE, Laniya TS, Buzas DM, Iturbe-Ormaetxe I, Carroll BJ, Gresshoff PM (2003) Long-distance signaling in nodulation directed by a CLAVATA1-like receptor kinase. Science 299:109–112. doi:10.1126/science.1077937
Shendure J, Ji H (2008) Next-generation DNA sequencing. Nat Biotechnol 26:1135–1145. doi:10.1038/nbt1486
Shure M, Wessler S, Fedoroff N (1983) Molecular identification and isolation of the Waxy locus in maize. Cell 35:225–233
Varshney RK, Nayak SN, May GD, Jackson SA (2009) Next-generation sequencing technologies and their implications for crop genetics and breeding. Trends Biotechnol 27:522–530. doi:10.1016/j.tibtech.2009.05.006
Wu J-L, Wu C, Lei C, Baraoidan M, Bordeos A, Madamba MRS, 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. doi:10.1007/s11103-004-5112-0
Zhang L, Fetch T, Nirmala J, Schmierer D, Brueggeman R, Steffenson B, Kleinhof A (2006) Rpr1, a gene required for Rpg1-dependent resistance to stem rust in barley. Theor Appl Genet 113:847–855. doi:10.1007/s00122-006-0342-y
Zhu M, Zhao S (2007) Candidate gene identification approach: progress and challenges. Int J Biol Sci 3:420–427
Acknowledgments
This study was supported by a Grant from the Next Generation BioGreen 21 Program (No. PJ00806003) of the Rural Development Administration, Republic of Korea. S.-H. L. and G. S. thank International Atomic Energy Agency 15664R2 and Grant #IOS1025752 from the Plant Genome Program, National Science Foundation, respectively.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Hwang, W.J., Kim, M.Y., Kang, Y.J. et al. Genome-wide analysis of mutations in a dwarf soybean mutant induced by fast neutron bombardment. Euphytica 203, 399–408 (2015). https://doi.org/10.1007/s10681-014-1295-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10681-014-1295-x