Rice Functional Genomics Project

Ac/Ds system

We have shown that Ac/Ds based gene and enhancer trap systems are suitable for generating insertion mutants in rice. The systems involves production of immobile Ac (iAc) and Ds (enhancer or gene trap) transgenic lines by Agrobacterium-mediated transformation, genetic crossing to produce mutagenic populations containing both, iAc and Ds and screening of plants from subsequent generations for stable (devoid of iAc) Ds insertions in new genomic locations and for any phenotypes associated with these insertions. Regions flanking the Ds element are being cloned and sequenced to create a database of flanking sequences that represent disrupted genes. Public sequence databases are being searched for homologues of these sequences. Among the 260 proven rice genomic sequences flanking Ds, 119 showed homology with published rice (Iinternational Rice Genome sequencing Program, Japan) or other (Natonal Centre for Biotechnology Information) known genes or sequences, including a submergence induced gene (Ossip2), several rice expansin genes, a rice adh, a rice MADS15 gene, Barley’s cysteine proteinase precursor and lipid transfer protein encoding genes, maize gene encoding thiamine biosynthetic enzyme, Arabidopsis genes encoding monooxygenase 1, cytosine-5-methyltransfe and the cotton sad1 gene. Almost all of our Ds flanking sequences are represented in the recently released China super hybrid rice genome sequence database. Genes or genetic regions responsible for acute dwarfism, early flowering, conditional sterility and several tissue specific gene expression profiles have also been identified and further studies are in progress here at Plant Industry and our collaborating laboratories.

New constructs
 

With the current tagging systems approximately 4% of any screening population are stable insertion lines which makes the screening very laborious and time consuming. We have now incorporated additional components in our iAc (immobile Ac)  and Ds constructs to facilitate high throughput screening.  Details will be provided shortly.

New tagging system
 

We have also explored the possibility of inducing transposition in callus cultures of proven Ds lines with an excision marker. This is achievable by transiently expressing transposase after co-cultivation with Agrobacterium harbouring an iAc construct containing gfp as a visual reporter gene and then selecting for excision⁺ GFP^- Ds⁺ regenerants.  Preliminary studies indicate that this transiently expressed transposase could induce Ds transposition. We are in the process of testing the suitability of this system for large-scale production of stable insertion lines from proven single locus Ds lines. Top

Bioinformatics

A labnote-cum-relational database has been created using Microsoft Access which catalogues Ac and Ds lines, Ds trap lines and flanking rice genomic sequences, mutant phenotypes, expression pattern, sequence information and sequence homology. A  “BLAST” searchable tagged sequence database has also been setup (http://www.pi.csiro.au/fgrttpub/blast_csn.htm). A web site is being designed for instant release information on lines with tags in known gene sequences (http://www.pi.csiro.au/fgrttpub/knowngene.htm).  A barcode system has been be implemented for tracking various lines, progenies, samples and other database entries. Bioinformatics capabilities of the group is being enhanced with the involvement of Centre for Mathematics and Information Science through NSW Agricultural Genomics Centre. A web-based Rice Gene Machine Information Management System (RGMIMS) is being developed.

International collaboration

Our efforts in hosting an international workshop Towards Building a Global Rice Gene Machine (http://grgm.pi.csiro.au/home.htm) in 2002 have led to the formation of the International Rice Functional Genomics Consortium (http://www.iris.irri.org/IRFGC/). We are one among 18 institutions (from 10 countries and two international agricultural research centres) represented on the Interim Steering Committee. The mandate of the IRFGC is to coordinate international rice functional genomics research and to build common strategies. We are taking an active part in this global initiative. Sharing our Gene Machine will allow us to access Gene Machines being developed by other members of this international consortium for identifying agronomically-important rice genes for further exploitation both in classical and transformation breeding.

In addition we are developing direct collaborations with researchers at five overseas laboratories (Brazil, Indonesia, Germany, Japan and India).

Group Members 
 

Current Members                                       
 

Narayana M Upadhyaya
Kerrie Ramm

Liz Dennis
Jim Peacock
 

Past Members and Visitors
 

Hui Wang, Jie Yang, Ramesh Bhat, Satya Nugroho, Marcia Margis, Kefan Peng, Tsuneo Kato (Visiting Scientists)
Andrew Eamens (PhD Student)
Xue Rong Zhou, Qian-Hao Zhu (PDFs)
Kathryn Smith, Shuting Pan, Limin Wu, Xueqin Wang, Weining Yu,  Ramani Sivakumar, Shamsul Hoque, Suzhi Li  (TO),
Dow-Won Yun, Song-Jin Kim, Song-Su Ma (Visitors)

Leaka Henry (Bioinformatics support, CMIS)

Publications
 

Refereed papers

Krishnan A, Guderdoni E, An G, Hsing Y-I, Han C-D, Lee MC, Yu S-M, Upadhyaya N, Ramachandran S, Zhang Q, Sundaresan V, Hirochika H, Leung H, Pereira A (2009) Mutant resources in rice for functional genomics of grasses. Plant Physiology 149:165-170.

Henry L,  Ramm K, Zhu Q-H, Upadhyaya NM (2008) RGMIMS - A web-based laboratory information management system for plant functional genomics research. Molecular Breeding 22:151-157

Xu, M, Ross Wilderman P, Morrone D, Xu J, Roy A, Margis-Pinheiro M, Upadhyaya NM, Coates RM, Peters RJ (2007) Functional characterization of the rice kaurene synthase-like gene family. Phytochemistry 68:312-326

Zhu Q-H, Ramm K, Eamens AL Dennis ES, Upadhyaya NM (2006) Transgene structures suggest that multiple mechanisms are involved in T-DNA integration in plants. Plant Science 171:308–322 (doi:10.1016/j.plantsci.2006.03.019)

Upadhyaya NM, Zhu Q-H, Zhou X-R, Eamens AL, Hoque MS, Ramm K, Shivakkumar R, Smith KF, Pan S-T, Li Suzhi, Peng K, Kim SJ, Dennis ES (2006)

Dissociation (Ds) constructs, mapped Ds launch pads and a transiently expressed transposase system suitable for localized insertional mutagenesis in rice. Theoretical and Applied Genetics 112:1326-1341(http://dx.doi.org/10.1007/s00122-006-0235-0).

Hoque MS, Masle J, Udvardi MK, Ryan PR and Upadhyaya NM (2006) Over-expression of the rice OsAMT1-1 gene increases ammonium uptake and content, but impairs growth and development of plants under high ammonium nutrition. Functional Plant Biology 33:153-163.

Islam N, Upadhyaya NM, Lonsdale M,  Higgins TJ, Campbell PM and Akhurst R (2005)  Decreased accumulation of glutelin types in rice grains constitutively expressing a sunflower seed albumin gene. Phytochemistry 66: 2534-2539.

Margis M, Zhou X-R, Zhu Q-H, Dennis ES and Upadhyaya NM (2005) Isolation and characterization of a Ds-tagged rice (Oryza sativa L.) GA responsive dwarf mutant defective in an early step of the gibberellin biosynthesis pathway. Plant Cell Reports 23:819-833.

Zhu  Q-H, Ramm K, Shivakkumar R, Dennis ES and Upadhyaya NM (2004) The ANTHER INDEHISCENCE1 gene encoding a single MYB domain protein is involved in anther development in rice (Oryza sativa L.). Plant Physiology 135:1514-1525.

Eamens AL, Blanchard CL, Dennis ES, and Upadhyaya NM (2004) A bidirectional gene trap construct for T-DNA and Ds mediated insertional mutagenesis in rice (Oryza sativa L.) Plant Biotechnology Journal 2:367-380.

Hirochika H, Guiderdoni E, An G, Hsing Y-I, Eun MY, Upadhyaya NM, Ramachandran S, Zhang Q, Pereira A, Sundaresan V and Leung  H (2004)  Rice mutant resources for gene discovery. Plant Molecular Biology 54:325-334.

Zhu Q-H, Hoque MS, Dennis ES and Upadhyaya NM (2003) Ds tagging of BRANCHED FLORETLESS 1 (BFL1) that mediates the transition from spikelet to floret meristem in rice (Oryza sativa L.). BMC Plant Biology online 3:6.

Upadhyaya NM, Zhou X-R, Ramm K, Zhu Q-H, L.Wu, Eamens A, Sivakumar R, Kato T, Yun D-W, Kumar S, Narayanan KK, Thomas G, Peacock WJ and Dennis ES (2002).  An iAc/Ds gene and enhancer trapping system for insertional mutagenesis in rice. Functional Plant Biology 29: 547-559. 

Upadhyaya NM, Zhou X-R, Wu L, Ramm K, Dennis ES (2000) The tms2 gene works as a negative selection marker in rice. Plant Molecular Biology Reporter 18:227-233.

Upadhyaya NM, Surin B, Schünmann P, Ramm K, Gaudron J, Taylor WC,Waterhouse PM  (2000) Agrobacterium-mediated transformation of Australian rice cultivars Jarrah and Amaroo with modified promoters and selectable markers. Australian Journal of Plant Physiology 27:201-210.

Other articles

Bhat RS, Upadhyaya NM, Chaudhury A, Raghavan C, Qiu F, Wang H, Wu J, McNally K, Leung H, Till B, Henikoff S and Comai L (2007) Chemical and irradiation induced mutants and TILLING. In: Upadhyaya NM (ed) Rice Functional Genomics- Challenges, Progress and Prospects. Springer, NY pp 149-180

Zhu Q-H, Eun MY, Han C-D, Kumar CS, Pereira A, Ramachandran S, Sundaresan V, Eamens AL, Upadhyaya NM and  Wu R (2007) Transposon insertional mutants: a resource for rice functional genomics. In: Upadhyaya NM (ed) Rice Functional Genomics- Challenges, Progress and Prospects. Springer, NY pp 223-271

Upadhyaya NM, Zhu Q-H and Dennis ES (2006) A gene machine for functional genomics of rice. A Report for Rural Industries Research and Development Corporation, Australia. RIRDC Publication No. W05/194 (downloadable from http://www.rirdc.gov.au/fullreports/rice.html )

Upadhyaya NM and Dennis ES (2005) New lines on offer at rice mutant gene library. IREC Farmers’Newsletter, 168, Rice R&D Edition: 48-49

Islam N, Lonsdale M, Upadhyaya NM, Higgins TJ, Hirano H, and Akhurst R (2004) Protein extraction from mature rice leaves for two-dimensional gel electrophoresis and its application in proteome analysis. Proteomics 4:1903-1908.

Eamens AL, Zhu Q-H, Dennis ES, Upadhyaya NM (2004) Designer constructs for T-DNA and Dissociation (Ds) mediated insertional mutagenesis in plants. ISB News Report November, 2004 http://www.isb.vt.edu/news/2004/news04.Nov.html

Upadhyaya NM, Zhou X-R, Zhu Q-H, Eamens A, Ramm K, Wu L, Sivakumar R, Kumar S, Narayanan KK, Thomas G, Kato T, Yun D-W, Peacock WJ, Dennis ES (2003). A gene machine for rice. In Khush GS, Brar DS, Hardy B (eds), Proceedings of the  4^th International Rice Genetics Symposium, 22-27 October 2000, Los Baños, Philippines: International Rice Research Institute. 352 p

Zhou X-R, Ramm K, Wu L, Sivakumar R, Dennis ES Upadhyaya NM (2003). New Ac/Ds-based constructs for efficient gene and enhancer trapping in rice. In Khush GS, Brar DS, Hardy B (eds), Proceedings of the  4^th International Rice Genetics Symposium, 22-27 October 2000, Los Baños, Philippines: International Rice Research Institute. 362 p.

Upadhyaya NM, Zhu Q-H, Eamens A and Dennis ES (2002) Rice gene machine: a vehicle for finding functions of cereal genes. Asia Pacific Biotech News 6:936-942.  

Upadhyaya NM, Zhou X-R, Zhu Q-H, Eamens A, Wang M-B, Waterhouse PM and Dennis ES (2000) Transgenic Rice. In Transgenic Cereals, eds O'Brien L , Henry RJ, AACC, Minnesota pp 28-87.

Invited/selected Presentations 

Abreu-Neto JB, Richter S, Bevitori R, Zanettini MH, Upadhyaya N, Margis-Pinheiro M (2009) Functional characterization of the gene OsGMBP1 of rice (Oryza sativa L.). Paper to be presented at the “II Simpósio Brasileiro de Genética Molecular de Plantas" (http://www.sbg.org.br/Eventos/IISBGMP/pt-br/index.html ).

Upadhyaya NM, Ramm K, Spriggs A (2008) Natural antisense transcripts (NATs), small RNAs and drought stress responsive gene control in rice. Proceedings 6^th International Symposium of Rice Functional Genomics (Jeju, Korea, 10-12 November, 2008

Upadhyaya NM, Ramm K, Spriggs A (2008) Naturally occurring antisense transcripts (NATs) of rice in stress responsive gene control. Poster presented at  COMBIO2008 (Canberra, Australia, 21-25 September 2008

Upadhyaya NM, Ramm K, Spriggs A (2007) Naturally occurring antisense transcripts (NATS) in stress responsive gene control in rice. Proceedings 5^th International Symposium of Rice Functional Genomics (Tsukaba, Japan, 15-17 October, 2007)

Upadhyaya NM, Zhu Q-H, Bhat RS, Hoque MS, Ramm K, Shivakkumar R, Dennis ES (2006) CSIRO launch pads for chromosomal region directed localized gene tagging in rice. Paper presented at the 4th International Symposium on Rice Functional Genomics, 9-11 October, 2006, Montpellier, France

Upadhyaya NM, Zhu Q-H, Bhat RS, Hoque MS, Ramm K, Shivakkumar R, Li S, Liu J, Dennis ES (2005) Mining Seedling Vigour Genes using the Rice Gene Machine. Poster presented at the 5 International Rice Genetics Congress 20-24 November 2005, Manila, Philippines.

Zhu Q-H, Dennis Es and Upadhyaya NM (2005) COMPACT SHOOT AND LEAFY HEAD1 (CSL1), a heterochronic gene  regulates leaf initiation and developmental transition in rice. Poster presented at the 5 International Rice Genetics Congress 20-24 November 2005, Manila, Philippines.

Henry L, Ramm K, Zhu Q-H and Upadhyaya NM (2005) Rice Gene Machine Information Management System (RGMIMS). Poster presented at the 5 International Rice Genetics Congress 20-24 November 2005, Manila, Philippines.

Bhat RS Zhu Q-H, Hoque MS, Ramm K, Shivakkumar R, Li S, Liu J, Dennis ES And Upadhyaya NM, 2005, Mining Seedling Vigour Genes using the Rice Gene Machine. Poster presented in COMBIO-2005 at Adelaide, Australia, 25-29, Sept, 2005. Abstract

Upadhyaya NM, Zhu Q-H, Hoque MS, Ramm K, Shivakkumar S, Smith K, Pan
S-T, Li S, Peng K, Dennis ES (2004) Dissociation (Ds) insertional mutagenesis using the transiently expressed transposase: Improved constructs and their suitability for targeted saturation mutagenesis. Invited paper presented at the 2nd International Symposium on Rice Functional Genomics held in Tucson, Arizona, USA (15-17 Nov. 2004).

Upadhyaya NM, Zhu Q-H, Eamens A, Margis M, Ramm K, Hoque MS, Shivakkumar R and Dennis ES (2003) Ac/Ds for insertional mutagenesis in rice Invited paper presented at the First International Symposium on Rice Functional Genomics held in Shanghai, China (19-21 Nov. 2003). Abstract    

Upadhyaya NM,  Zhu Q-H, Eamens A, Dennis E (2003) Rice Gene Machine – A key to finding functions of cereal genes. Symposium paper presented at the XIX International Congress of Genetics, Melbourne, Australia (6-11 August 2003) (Abstract)

Upadhyaya NM, Zhou X-R, Zhu Q-H, Eamens A, Peacock J and Dennis ES (2001). Rice gene machine. Invited presentation at the "Functional Genomics workshop" held as a part of "International Rice Genome Meeting 2001, Tsukuba, Ibaraki (7-9 Feb. 2001).

Upadhyaya NM, Kumar S, Ramm K, Wu L,  Zhou X-R Narayanan K, Thomas G, Waterhouse PM, Chaudhury A, Peacock J, Dennis E. Functional genomics in rice by transposon tagging (1999)  Paper presented at the General Meeting of the International Program on Rice Biotechnology, held at Phuket, Thailand, 20-24 Sept 1999.

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