Abstract
Key message
Novel drought tolerance genes were identified by screening thousands of random genomic fragments from grass species in transgenic rice.
Abstract
Identification of agronomically important genes is a critical step for crop breeding through biotechnology. Multiple approaches have been employed to identify new gene targets, including comprehensive screening platforms for gene discovery such as the over-expression of libraries of cDNA clones. In this study, random genomic fragments from plants were introduced into rice and screened for drought tolerance in a high-throughput manner with the aim of finding novel genetic elements not exclusively limited to coding sequences. To illustrate the power of this approach, genomic libraries were constructed from four grass species, and screening a total of 50,825 transgenic rice lines for drought tolerance resulted in the identification of 12 reproducibly efficacious fragments. Of the twelve, two were from the mitochondrial genome of signal grass and ten were from the nuclear genome of buffalo grass. Subsequent sequencing and analyses revealed that the ten fragments from buffalo grass carried a similar genetic element with no significant homology to any previously characterized gene. The deduced protein sequence was rich in acidic amino acid residues in the C-terminal half, and two of the glutamic acid residues in the C-terminal half were shown to play an important role in drought tolerance. The results demonstrate that an open-ended screening approach using random genomic fragments could discover trait genes distinct from gene discovery based on known pathways or biased toward coding sequence over-expression.
Similar content being viewed by others
References
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815
Benfey PN, Chua NH (1990) The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants. Science 250:959–966
Bor NL (1960) The grasses of Burma, Ceylon, India and Pakistan (excluding Bambuseae). Pergamon Press, London
Chou CC, Wang AH (2015) Structural D/E-rich repeats play multiple roles especially in gene regulation through DNA/RNA mimicry. Mol BioSyst 11:2144–2151
Christensen AH, Sharrock RA, Quail PH (1992) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol 18:675–689
Dinesh-Kumar SP, Baker BJ (2000) Alternatively spliced N resistance gene transcripts: their possible role in tobacco mosaic virus resistance. Proc Natl Acad Sci USA 97:1908–1913
Doebley JF, Gaut BS, Smith BD (2006) The molecular genetics of crop domestication. Cell 127:1309–1321
Draper CK, Hays JB (2000) Replication of chloroplast, mitochondrial and nuclear DNA during growth of unirradiated and UVB-irradiated Arabidopsis leaves. Plant J 23:255–265
El Baidouri M, Carpentier MC, Cooke R, Gao D, Lasserre E, Llauro C, Mirouze M, Picault N, Jackson SA, Panaud O (2014) Widespread and frequent horizontal transfers of transposable elements in plants. Genome Res 24:831–838
Fukayama H, Tsuchida H, Agarie S, Nomura M, Onodera H, Ono K, Lee BH, Hirose S, Toki S, Ku MS, Makino A, Matsuoka M, Miyao M (2001) Significant accumulation of C4-specific pyruvate, orthophosphate dikinase in a C3 plant, rice. Plant Physiol 127:1136–1146
Habben JE, Bao X, Bate NJ, DeBruin JL, Dolan D, Hasegawa D, Helentjaris TG, Lafitte RH, Lovan N, Mo H, Reimann K, Schussler JR (2014) Transgenic alteration of ethylene biosynthesis increases grain yield in maize under field drought-stress conditions. Plant Biotechnol J 12:685–693
Hiei Y, Komari T (2008) Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat Protoc 3:824–834
Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282
Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180
Ichikawa T, Nakazawa M, Kawashima M et al (2006) The FOX hunting system: an alternative gain-of-function gene hunting technique. Plant J 48:974–985
International Rice Genome Sequencing Project (2005) The map-based sequence of the rice genome. Nature 436:793–800
International Wheat Genome Sequencing Consortium (2018) Shifting the limits in wheat research and breeding using a fully annotated reference genome. Science 361:eaar7191
ISAAA (2018) Global status of commercialized biotech/GM crops in 2018: biotech crops continue to help meet the challenges of increased population and climate change. ISAAA Brief No. 54. ISAAA, Ithaca, NY
Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750
Ishida Y, Tsunashima M, Hiei Y, Komari T (2015) Wheat (Triticum aestivum L.) transformation using immature embryos. Methods Mol Biol 1223:189–198
Kaiser K, Murrey N (1985) Construction of representative genomic DNA libraries. In: Glover DM (ed) DNA cloning, vol 1. IRL Press Ltd., Oxford, pp 1–47
Kondou Y, Higuchi M, Takahashi S et al (2009) Systematic approaches to using the FOX hunting system to identify useful rice genes. Plant J 57:883–894
Lawrence JG, Roth JR (1996) Selfish operons: horizontal transfer may drive the evolution of gene clusters. Genetics 143:1843–1860
Lee K, Zhu H, Yang B, Wang K (2019) An Agrobacterium-mediated CRISPR/Cas9 platform for genome editing in maize. Methods Mol Biol 1917:121–143
Lemos ML, Crosa JH (1992) Highly preferred site of insertion of Tn7 into the chromosome of Vibrio anguillarum. Plasmid 27:161–163
Liu L, White MJ, MacRae TH (1999) Transcription factors and their genes in higher plants functional domains, evolution and regulation. Eur J Biochem 262:247–257
Liu G, Campbell BC, Godwin ID (2014) Sorghum genetic transformation by particle bombardment. In: Henry R, Furtado A (eds) Cereal genomics: methods in molecular biology (methods and protocols). Humana Press, Totowa, pp 219–234
Mann HB, Whitney DR (1947) On a test of whether one of two random variables is stochastically larger than the other. Ann Math Stat 18:50–60
Miousse IR, Chalbot MC, Lumen A, Ferguson A, Kavouras IG, Koturbash I (2015) Response of transposable elements to environmental stressors. Mutat Res Rev Mutat Res 765:19–39
Moyers BT, Morrell PL, McKay JK (2018) Genetic costs of domestication and improvement. J Hered 109:103–116
Nakamura H, Hakata M, Amano K et al (2007) A genome-wide gain-of function analysis of rice genes using the FOX-hunting system. Plant Mol Biol 65:357–371
Nuccio ML, Wu J, Mowers R, Zhou HP, Meghji M, Primavesi LF, Paul MJ, Chen X, Gao Y, Haque E, Basu SS, Lagrimini LM (2015) Expression of trehalose-6-phosphate phosphatase in maize ears improves yield in well-watered and drought conditions. Nat Biotechnol 33:862–869
Nuccio ML, Paul M, Bate NJ, Cohn J, Cutler SR (2018) Where are the drought tolerant crops? An assessment of more than two decades of plant biotechnology effort in crop improvement. Plant Sci 273:110–119
Qu R, Luo H, Meier VD (2008) Turfgrass. In: Kole C, Hall TC (eds) Compendium of transgenic crop plants: transgenic plantation crops, ornamentals and trufgrasses. Blackwell Publishing, Oxford, pp 177–218
Reuzeau C, Frankard V, Hatzfeld Y, Sanz A, Van Camp W, Lejeune P, De Wilde C, Lievens K, de Wolf J, Vranken E, Peerbolte R, Broekaert W (2006) Traitmill™: a functional genomics platform for the phenotypic analysis of cereals. Plant Genet Resour 4:20–24
Sainz MB, Goff SA, Chandler VL (1997) Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo. Mol Cell Biol 17:115–122
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York
Schnable PS, Ware D, Fulton RS et al (2009) The B73 maize genome: complexity, diversity, and dynamics. Science 326:1112–1115
Xiong Y, Eickbush TH (1990) Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J 9:3353–3362
Yin K, Gao C, Qiu JL (2017) Progress and prospects in plant genome editing. Nat Plants 3:17107
Yu C, Wang L, Xu S, Zeng Y, He C, Chen C, Huang W, Zhu Y, Hu J (2015) Mitochondrial ORFH79 is essential for drought and salt tolerance in rice. Plant Cell Physiol 56:2248–2258
Zhang Y, Malzahn AA, Sretenovic S, Qi Y (2019) The emerging and uncultivated potential of CRISPR technology in plant science. Nat Plants 5:778–794
Acknowledgements
The authors thank the members of our research institutes for valuable discussions and technical assistance.
Accession numbers
pLC20GWH, LC506529; pLC31GWH, LC506530; HF001, LC507188; HF003, LC507189; HF050, LC507217; HF051, LC507218.
Author information
Authors and Affiliations
Contributions
TKomo, YS, NK, NT, NB and TKoma designed the research and experiments. TKomo, YS, MK, NU, SU, NI, YH, KK, RK, EB and NB performed the experiments. TKomo, YS, KW, PO, NT, NB and TKoma regularly discussed the research progress and developed the research strategy. TKomo, YS, NB and TKoma wrote the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
All authors are or were affiliated with Japan Tobacco Inc. or Syngenta Crop Protection LLC, who were also funders of this study.
Additional information
Communicated by Lizhong Xiong.
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.
Rights and permissions
About this article
Cite this article
Komori, T., Sun, Y., Kashihara, M. et al. High-throughput phenotypic screening of random genomic fragments in transgenic rice identified novel drought tolerance genes. Theor Appl Genet 133, 1291–1301 (2020). https://doi.org/10.1007/s00122-020-03548-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00122-020-03548-6