Faculty and research guide

Laboratory Genetics and Genomics

Motonori Tomita

The Laboratory Genetics and Genomics manages an array of genetics, genomics, and molecular biology approaches to isolate novel genes associated with higher tolerance to environmental stress and consequently higher yields from plant genetic resources, while investigating the functions of these genes. We then apply these findings to develop novel cultivars based on the genomics-assisted breeding. Our work has been published in international journals in the fields of genetics, genomics, biotechnology, evolution, cytogenetics, botany, and breeding science, and our innovations such as the novel genes and cultivars have been patented. We are currently conducting projects in two groups within the Research Institute of Green Science and Technology (http://www.green.shizuoka.ac.jp/green_bio_intro.html), namely the Green Biology Research Division and the Functional Genomics Section (http://www.shizuoka.ac.jp/~idenshi/).

I. Genome-specific transposons in Triticeae
Plant resources closely related to wheat, such as rye and Thinopyrum, tolerate unfavorable environmental conditions and have large genomes that have transposons as the main components. We considered that transposons which can move and multiply in the specific genome would serve as useful DNA markers and tools, such as mutagens, in molecular breeding. In addition, we have successfully isolated the transposon-like elements Revolver and Superior, which are unique in closely related genomes. We first isolated rye-specific sequences via the genomic subtraction of common DNA sequences between rye and wheat and then constructed a library containing long DNA fragments to enable sequencing of these rye-specific fragments. A transposon with a completely novel structure, which therefore does not belong to any known category of transposable elements, was discovered and named Revolver (evolution-inducing element). Further, transposase-like cDNA and non-autonomous elements of Revolver were isolated, and its considerable genetic divergence was revealed by bioinformatics analysis. The Revolver transposon exists at a high copy number (10,000) in rye and has been found in ancestral species of wheat, but current generations of wheat appear to have lost Revolver during evolution. We also found considerable quantitative differences in the Revolver copy number among first-generation crossbreeds of cultivated wheat and various populations grown under a wide range of ecological conditions in Israel, which is located close to where wheat originated. These results suggest that Revolver is transposable and contributes to genomic divergence.
Meanwhile, we achieved efficient isolation of novel transposable elements using EcoO109I, which recognizes and cleaves multiple sites. We further developed methods to enable transposons that specifically exist in species closely related to wheat, but not in wheat itself, to be utilized as DNA markers for fluorescence in situ hybridization. We inserted genes into genomes using chromosome engineering technology, and we identified genomes containing newly inserted genes mediating tolerance to biological and environmental stress. We have organized the Transposon Symposium in Japanese Society of Breeding.

II. Isolation and pyramiding of useful genes in rice using a genomics approach
The introduction of semidwarf cereal cultivars has enabled the Green Revolution, but a single semidwarf gene, sd1, is solely responsible for semidwarf phenotypes in world rice. Thus, we examined mutants to identify novel semidwarf genes based on the fact that the semidwarf trait does not segregate in accordance with Mendel’s laws and is associated with spike sterility in F2 progenies. We successfully discovered d60, a new potentially useful semidwarf gene that complements the gametic lethal gene gal, and we revealed its unique mode of inheritance, wherein pollens carrying both d60 and gal acquire gametic lethality through the disappearance of the vegetative nucleus after the first divisions of the generative nucleus. We also found that both japonica and indica varieties commonly carry gal, and thus the mutation of gal to Gal, which does not occur naturally, was essential for the inheritance of d60. We then localized d60 and Gal on chromosome 2 and chromosome 5, respectively, by gene mapping using DNA markers. We isolated d60 by map-based cloning and revealed the function of d60 by a functional complementation test with genetic engineering techniques. Meanwhile, we conducted genomics-assisted breeding approaches to introduce sd1 or d60 into the main rice variety Koshihikari, which has poor lodging resistance, and we successfully developed short-culm Koshihikari, namely Hikarishinseiki and Minihikari (both granted under the Japan Plant Variety Protection (PVP) by the Ministry of Agriculture, Forestry and Fisheries). Hikarishinseiki is a certified brand variety of the growing district in fourteen prefectures, including Niigata Prefecture, and was the first short-culm Koshihikari announced in a press release by the Japanese Society of Breeding. We also introduced late maturing genes into the short-culm Koshihikari in our JST-funded anti-global warming project. The PVP registration application of the novel variety is currently pending.

On the basis of these findings, we are currently conducting research into the following areas.

(1) Isolation of novel genes responsible for large grain, short culm, and desirable maturing characteristics, and the accumulation of these genes as measures against global warming and market globalization.
The Japanese agricultural industry is facing its toughest situation today. The agricultural environment is deteriorating because of global warming and post-earthquake damage such as salt and radioactive contamination. Moreover, the opening of Japan’s agricultural product market after joining the Trans-Pacific Partnership will accelerate the import of Koshihikari from USA and China. To overcome such situations, it is crucial to develop new cultivars superior to Koshihikari, such as those that can adapt to deteriorating conditions and possess novel traits including low cost and high yielding.
The Laboratory of Genetic Engineering examines mutants by genomics and genetic engineering approaches to discover useful genes that are responsible for favorable traits, such as those that improve cost efficiency, yield (high lodging resistance, large grains, and high biomass), adaptability (maturing characteristics), and stress resistance (heat tolerance during grain ripening and salinity resistance), and to elucidate their mechanisms. We then accumulate these genes by genomics-assisted breeding, in order to develop novel plant cultivars in the new era. As a measure against radioactive pollution, we employ genomics-assisted breeding approaches to develop super-early-maturity rice varieties that can be harvested four times per year in a plant factory. Moreover, to address the global food crisis, we aim to develop japonica varieties that can be grown in low-latitude areas, such as in Cambodia.
With the aim of achieving social contribution, we conduct genomics research and breeding biotechnology approaches to develop new cultivars. This is the green innovation that leads to advances in agriculture.

(2) Functional genomics research by focusing on novel transposons
Transposons are the main components of plant genomes and play an important role in genomic variation. We are interested in novel transposons in relation to genome function. They generate various noncoding RNAs, thereby may be regulating gene expression, vital phenomena such as ontogenesis, and evolution. We then try to isolate miRNAs transcribed from these transposons and revealed their target genes, as well as the effect of RNAi knockdown on phenotypic expression. We also try to understand their kinetics within a certain locus among species. We have also employed transposon display to create DNA markers as genetic engineering tools in functional genomics approaches.
(3) Isolation and functional analysis of genes responsible for tolerance to biological and environmental stress
We perform transcriptome and expression profile analyses to detect and identify novel alien genes involved in tolerance to biological and environmental stress, such as nematode damage and salt damage. We then try transgenic analysis to reveal the functions of these novels genes.
(4) Dynamics of plant genomes under environmental stress and during speciation
We examine transposon expression in plants those with considerable genetic variation that adapt to severe conditions, such as arid land, swamp, and cold area, and those produced in the course of the development of new species by genomic polyploidization. We then investigate the relationships of transposon and environmental stress or combination of genomes, and speciation.



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[Research achievements in the last five years]
Naito, Y., and Tomita, M.: Identification of an isogenic semidwarf rice cultivar carrying the Green Revolution sd1 gene using multiplex codominant ASP-PCR and SSR markers. Biochemical Genetics 51:DOI 10.1007/s10528-013-9584-y (2013)
Ma, Y.-Z., and Tomita, M.: Thinopyrum 7Ai-1-derived small chromatin with barley yellow dwarf virus (BYDV) resistance gene integrated into the wheat genome with retrotransposon. Cytology and Genetics 47(1):1-7 (2013)
Tomita, M.: Combining two semidwarfing genes d60 and sd1 for reduced height in 'Minihikari', a new rice germplasm in the 'Koshihikari' genetic background. Genetics Research Cambridge 94(5):235-244 (2012)
Tomita, M.: Recent patent on Revolver-2: a novel transposon-like gene useful for chromosome tags of rye. In New Developments in Chromatin Research (Eds. Neil M. Simpson and Valerie J. Stewart, ISBN 978-1-62081-816-9), Nova Science Publishers, New York, pp.161-176 (2012)
Tomita, M., and Seno, A.: Rye chromosome-specific polymerase chain reaction products developed by primers designed from the EcoO109I recognition site. Genome 55(5):370-382 (2012)
Tomita, M., and Matsumoto, S.: Transcription of rice Green Revolution sd1 gene is clarified by comparative RNA diagnosis using the isogenic background. Genomics and Applied Biology 2(5):29-35, doi: 10.5376/gab.2011.02.0005 (2011)
Tomita, M., Okutani, A., Beiles, A., and Nevo, E.: Genomic, RNA, and ecological divergences of the Revolver transposon-like multi-gene family in Triticeae. BMC Evolutionary Biology 11:269, doi:10.1186/1471-2148-11-269 (2011)
Tomita, M., and Tanisaka, T. Long-culm mutations with dominant genes are induced by mPing transposon in rice. Hereditas 147(6):256-263 (2010)
Tomita, M., Asao, M. and Kuraki, A. Effective isolation of retrotransposons and repetitive DNA families from the wheat genome. Journal of Integrative Plant Biology 52(7):679-691 (2010)
Tomita, M. and Misaki, M. KpnI-repetitive DNA element tandemly clustered on subtelomeric regions of Triticeae genome. Caryologia 63(1):91-98 (2010)
Tomita, M. Revolver and Superior: novel transposon-like gene families of the plant kingdom. Current Genomics 11(1):62-69 (2010)
Tomita, M., Noguchi, T. and Kawahara, T. Quantitative variation of Revolver transposon-like genes in synthetic wheat and their structural relationship with LARD element. Breeding Science 59(5):629-636 (2009)
Tomita, M. Introgression of Green Revolution sd1 gene into isogenic genome of rice super cultivar Koshihikari to create novel semidwarf cultivar 'Hikarishinseiki' (Koshihikari-sd1). Field Crops Research 114(2):173-181 (2009)
Tomita, M., Kuramochi, M. and Iwata, S. Superior: a novel repetitive DNA element dispersed in the rye genome. Cytogenetic and Genome Research 125(4):306-320 (2009)
Tomita, M., Akai, K. and Morimoto, T. Genomic subtraction recovers rye-specific DNA elements enriched in the rye genome. Molecular Biotechnology 42(2):160-167 (2009)
Yuan, W.-Y. and Tomita, M. Centromeric distribution of 350-family in Dasypyrum villosum and its application to identifying Dasypyrum chromatin in the wheat genome. Hereditas 146(2):58-66 (2009)
Tomita, M., Shinohara, K. and Morimoto, M. Revolver is a new class of transposon-like gene composing the Triticeae genome. DNA Research 15(1):49-62 (2008)

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