Indian Agricultural Research Institute New Delhi 110012 National Research Centre on Plant Biotechnology

Course: MOLECULAR GENETICS (MBB-505)

ASSIGNMENT Genomic Resources for Insertional Mutagenesis in Few Plants.

Submitted by Donald James MBB Roll No: 20078

INTRODUCTION Insertional mutagenesis is the use of DNA sequences as mutagens. A number of different insertional mutagenesis systems have been developed for plants, but all are based either on transposons or the integration of T-DNA. A number of transposons have been used for mutagenesis programs, including Activator (Ac), Suppressor-mutator (Spm) and Mutator (Mu) from corn (Zea mays; maize), Tam3 from Antirrhinum majus, and Tos17 from rice (Oryza sativa). Some of these transposons function only in their natural host plant, while others are promiscuous and can be used in a variety of heterologous species. The corn Ac element, for example, has been shown to function in Arabidopsis, tobacco, rice, potato (Solanum tuberosum), petunia (Petunia hybrida), tomato (Lycopersicon esculentum), and many other plants.T-DNA is not a transposon but is a short segment of DNA that is transferred from a bacterial plasmid to the plant genome when plants are infected by the soil pathogen Agrobacterium tumefaciens. T-DNA transfer by A. tumefaciens has been used extensively to generate transgenic plants and more recently, for insertional mutagenesis. The most direct way to establish the function of a plant gene is by mutation. Traditionally, genefunction relationships have been studied in a phenotype driven approach, sometimes described as forward genetics. Mutagenesis has been achieved by irradiating seeds or treating them with chemical mutagens, and then searching for interesting mutant phenotypes in subsequent generations. If a large enough population of seeds is mutagenized, it should be possible to recover mutants affecting any gene in the genome The disadvantage of radiation and chemical mutagens is that they tend to generate point mutations, which are difficult to map and make the process of gene isolation laborious and time consuming. Insertional mutagenesis is advantageous because genes tend to be completely disrupted, which favors the recovery of null mutants. Furthermore, the interrupted gene becomes tagged with the insertion, which can be used to clone flanking sequences, either by traditional means or strategies based on the polymerase chain reaction (PCR). The approach is generally known as gene tagging or signature tagged mutagenesis (STM). Reverse genetics is a gene-driven approach, in which a sequence with no known function is deliberately mutated allowing the resulting mutant phenotype to be studied Insertional mutagenesis has recently become established as an alternative reverse genetics strategy in plants. Although it is not possible to systematically target specific genes for disruption, it is possible to saturate the genome with mutations and maintain a resource of seeds representing interruptions in every single gene. A researcher discovering a particular gene and wanting to know its function can then search for insertional mutants affecting the gene of interest, either by screening the mutant seed bank or by searching databases of flanking sequences.

Thus of late the importance of Genomic resources such as databases on mutant seed stocks of various crops and plant species have become an important part of the functional genomics and molecular genetic studies of plants. This assignment provides a glimpse into the various genomic resources for insertional mutagenesis available for a few major plant species viz. Arabidopsis, Maize, Rice and Tomato .The World Genetic Resource site under the Shared information on Genetics resources(SHIGEN) maintained by the SHIGEN project carried out at the Center for Genetic Resource Information, National Institute of Genetics, Mishima, Japan gives a glimpse of all such resources and their corresponding links.
http://www.shigen.nig.ac.jp/wgr/quickSearchAction.do

Genomic resources for insertional mutagenesis in Arabidopsis. Insertional mutagenesis techniques, including transposon and TDNAmediated mutagenesis, are key resources for systematic identification of gene function in the model plant species Arabidopsis thaliana. Researchers at The Cold Spring Harbor Lab, NY, USA along with the John Innes Centre, Norwich, UK have developed a database called the ATIDB: Arabidopsis thaliana insertion database (http://atidb.cshl.org/) for archiving, searching and analyzing insertional mutagenesis lines. Flanking sequences from approximately 10 500 insertion lines (including transposon and TDNA insertions) from several tagging programs in Arabidopsis were mapped to the genome sequence through our annotation system before being entered into the database. (Xiaokang Pan et al Nucl. Acids Res. (2003) 31 (4):12451251.doi: 10.1093/nar/gkg222) Some important links which give access to all genomic resources of Arabidopsis are TAIR, The Arabidopsis Information Resource http://www.arabidopsis.org/ GARNet . An Arabidopsis thaliana Genomic Reource collaboration for UK based scientists.Its resources for Mutants collections in Arabidopsis can be found at http://www.garnetcommunity.org.uk/resources/mutant-collections ATIDB : The Arabidopsis thaliana Integrated Database available at http://atidb.org/

Some important Stock centers and mutant lines have been listed below with references. Worldwide Forward and Reverse Genetics Resources for Arabidopsis. (Insertional lines
Resource name Backgroun Vector Info d Line Col-0 pROK2 Availab le From

Type

View Mutation on Genome

Alonso, Crosby Simple insert (sets and Ecker of pools and individual lines)

n/a

ABRC NASC

Biological Promoter trap and Research activation tag Center(Hungar (individual lines) y)* CSHL Gene trap and enhancer trap (individual lines) Gene trap transposon (individual lines) Simple insert (sets of pools and individual lines) T-DNA insertion (individual lines) Single insert DsSpm (insertion lines) Ds insertion (individual lines)

Col

multiple TDNA vectors

locus mapping available on project website

contact authors

Ler

Ac, DsG and DsE T-DNAs

AtEnsemblAtIDBSeqViewerS ABRC IGnAL NASC

EXOTIC/ JIC Gene Trap

Ler

Ds-GT and Tpase

AtEnsemblAtIDBSIGnAL

ABRC NASC

Feldmann

Ws-2 3850:1003 Ti

n/a

ABRC NASC

GABI-Kat *

Col

pAC161

AtEnsemblAtIDBSeqViewerS ABRC IGnAL NASC AtEnsemblAtIDBSIGnAL ABRC NASC

GARNet - JIC SM

Col-0 SLJ8313 SLJ8337 SLJ8353 Ler

IMA *

Ds element SeqViewerSIGnAL (kanamycin resistance, GUS reporter) Tn113 (Ds starter line) X Tn25 (Ac transposase lines) n/a AtEnsemblAtIDB

ABRC NASC

JIC Activate

Activation trap (individual lines)

Ler

ABRC NASC

Misc. Insertion Misc. Collections Misc. T-DNA pools for forward screening Misc.

Misc.

n/a

ABRC NASC ABRC NASC

Misc.

n/a

n/a

RIKEN *

Ds transposon insertion (individual lines))

No-O series of Ds constructs

AtEnsemblRARGESIGnAL

RIKEN BRC

SAIL (formerly T-DNA insertion GARLIC/TMRI (individual lines) ) SALK T-DNA insertion (individual lines)

Col

pCSA110pDAP AtEnsemblAtIDBSeqViewerS ABRC 101 IGnAL NASC

Col

pROK2

AtEnsemblAtIDBSeqViewerS ABRC IGnAL NASC SeqViewerSIGnAL ABRC NASC

Confirmed T-DNA insertion Col SALK insertion (PCRlines validatedhomozygo us or heterozygous individual lines) Sussman and Amasino TAMARA

pROK2

Simple insert (set of Ws-2 pD991-AP3 pools) Tn mediated activation tag (individual lines) Col-0 TAMARA

n/a

ABRC NASC NASC

contact authors

Wisconsin Ds- Ds-Lox insertion Lox (individual lines) Wisconsin KO T-DNA insertion (set of pools)

Col

pDS-Lox

AtEnsemblAtIDBSeqViewerS ABRC IGnAL NASC n/a ABRC NASC

Ws

pD991-AP3

The above mentioned stock centers are 1) The Arabidopsis Biological Resource Center (ABRC) maintained by the Ohio State University, USA. Details of all mutant and mapping populations can be seen by searching the Arabidopsis Information Resource 2) The Nottingham Arabidopsis Stock Centre (NASC) also known as The European Arabidopsis Stock Centre provides seed and information resources to the International Arabidopsis Genome Programme and the wider research community and is maintained at the University of Nottingham, Sutton Bonington Campus, and UK.Details on various lines of mutants at the NASC can be seen by following the link http://arabidopsis.info/BrowsePage 3) Another database and stock centre is the RIKEN Plant Science centre of the Yokohama Institute, Japan ( RIKEN Bioresource Center (BRC)/ SENDAI Arabidopsis Seed Stock Center (SASSC) )The details can be found at RARGE-Riken Arabidopsis Genomic Encyclopedia http://rarge.psc.riken.jp/dsmutant/index.pl or http://www.brc.riken.jp/lab/epd/Eng/catalog/seed.shtml 4) INRA-Versailles Genomic Resource Center located at IJPB Versailles, France has also a collection of Arabidopsis T-DNA insertional mutant lines called AGROBACT + . Details can be availed at http://www-ijpb.versailles.inra.fr/en/cra/cra_accueil.htm or http://dbsgap.versailles.inra.fr/agrobactplus/English/Accueil_eng.jsp 5) GABI - Genome Analysis of the Plant Biological System from the Max-Planck-Institute for Molecular Plant Physiology,Germany.

The important Insertional mutant lines include 1. Salk T-DNA lines provided by the SALK Institute, La Jolla, CA USA. [About Salk T-DNA] 2. SAIL T-DNA lines. Syngenta Arabidopsis Insertion Library (SAIL) collection [About the SAIL lines] [Order from ABRC ]. 3. GABI-Kat lines GABI - Genome Analysis of the Plant Biological System from the MaxPlanck-Institute for Molecular Plant Physiology ,Germany. 4. FLAGdb/FST of the Institute of Agronomic Research, Versailles, France ( INRA Versailles Stock ) 5. Wisconsin T-DNA lines provided by the University of Wisconsin ,Madison USA. 6. CSHL genetrap lines provided by the Cold Spring Harbor Lab ,NY,USA. 7. RIKEN T-DNA & Ds lines. [Order from RIKEN BRC]( RIKEN / SENDAI SASSC stock) 8. JIC SM lines. [About the SM lines] [NASC] from the Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom, 9. IMA Ds lines from Institute of Molecular Agrobiology, National University of Singapore. 10. Saskatoon SK Lines provided by the Agriculture and Agri Food Department of Canadian Govt.

Details of Accessions

Salk T-DNA Salk T-DNA Homozygous SAIL FST SAIL T-DNA Homozygous Wisc FST Columbia

137,259 lines 33,744 lines

166,127 mapped 40,652 mapped 62,041 mapped

10/28/2009 05/18/2011 10/28/2009 05/18/2011 09/30/2009

4,522 lines 32,607

4,828 mapped 21,406 mapped

(22,089) Wisc Homozygous by Salk FLAGdb FST GABI-Kat FST GABI-Kat Confirmed GABI-Kat Homozygous by Salk RIKEN FST JIC SM FST Landsberg N^ssen 22,664 w seeds 24,444 (17,689 lines) 488 lines Col-4 15,507 lines Columbia Wassilevskija 1,981 lines 41,369 130,654 7,656 lines 1,040 lines 1,981 mapped 37,142 mapped 05/18/2011 06/23/2009

98,559 mapped (70,578 06/29/2011 lines) 11,030 mapped 1,373 mapped 18,622 mapped 24,206 mapped 04/12/2010 05/18/2011 06/23/2009 10/28/2009

CSHL FST IMA FST Saskatoon FST Other FST Total

23,998 mapped 808 mapped 14,941 mapped 175 mapped 452,065 mapped

10/25/2010 10/28/2009 08/17/2009 06/23/2009 12/01/2010

References 1.) T-DNA insertional mutagenesis in Arabidopsis: a tool for functional genomics Radhamony R.N,Ramamurthy Srinivasan et al, Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.8 No.1, Issue of April 15, 2005

Genomic resources for insertional mutagenesis in Rice. Various international groups are working to produce and characterize diverse rice mutant stocks. The table below provides links to such resources: Hirohiko Hirochika NIAS, National Institute of Agrobiological Sciences, Japan. Postech Plant Functional Genomics Laboratory Korea CSIRO Plant Industry Rice Functional Genomics Group Australia OrygenesDb, OryzaTagLines Tos17 Insertion Mutant Database T-DNA Insertion Mutants T-DNA and Ds Insertion Mutants

Gyn An

Narayana Upadhyaya Emmanuel Guiderdoni

Insertional mutant

Group

Alias

Publicatio n [View]

Contact

Vector

Number of FST 611

CSIRO National Institute of Agrobiological Sciences CerealGene Tags, European Union CIRAD-INRA-IRDCNRS,Genoplante CIRAD-Genoscope Gyeongsang National University

CSIRO

narayana.upadhyaya(@)csi T-DNA ro.au hirohiko(@)nias.affrc.go.jp Tos17

NIAS

[View]

18,024

OSTID OTL TDNA OTL Tos17

[View]

andy.pereira(@)wur.nl

Ds

1,380

[View]

emmanuel.guiderdoni(@)ci T-DNA rad.fr emmanuel.guiderdoni(@)ci Tos17 rad.fr cdhan(@)nongae.gsnu.ac.k Ds r

27,555

14,355

PMBBRC [View]

1,072

Plant Functional Genomics Laboratory

Postech [View]

genean(@)postech.ac.kr

T-DNA 107,171

National Center of Plant RMD Gene Research (Wuhan) Shanghai T-DNA Insertion Population Taiwan Rice Insertional Mutant Program SHIP

[View]

swang(@)mail.hzau.edu.cn T-DNA

33,197

ship(@)sibs.ac.cn

T-DNA

12,614

TRIM

[View]

bohsing(@)gate.sinica.edu.t T-DNA w sundar(@)ucdavis.edu Ds

11,799

University of California at UCD Davis Total References 1.)OrygenesDB website 2.)Oryzabase website

[View]

17,730 245,508

Genomic resources for insertional mutagenesis in Maize.

The RescueMu Maize Mutant Phenotype Database available at the MaizeGDB (Maize Genetics and Genomics Database ).This phenotype database is part of the NSF project "Maize Gene Discovery, DNA Sequencing, and Phenotypic Analysis." A large population of maize plants was generated aiming to contain RescueMu transposon insertions in every maize gene. These plants were screened for morphological abnormalities and the phenotype observations were recorded in this database. Corresponding seeds can be obtained from the Maize Genetics Cooperation - Stock Center. The Maize Gene Discovery Project is sequencing maize DNA flanking RescueMu transposons, and provides this information and plants (identified by a PCR-based assay) that contain the mutant allele of interest to users for a fee of $150. UniformMu is a unique maize population developed for maize genetics research (McCarty et al., 2005). This material is widely used for experimental analysis of maize gene function, because 1) the uniform plants and seeds provide an excellent basis for comparing unaltered, wild-type controls to detectable changes in new plant phenotypes, 2) the UniformMu transposable elements actively generate new mutations when they insert into individual genes, and 3) a searchable database (at MaizeGDB.org) matches gene sequences to specific mutants with Mu insertions in genes of interest (with seed stocks immediately available from the Maize Genetics Cooperation Stock Center via MaizeGDB.org)Links http://endosperm.info/, http://uniformmu.org) The Maize Targeted Mutagenesis population is publically available (http://mtm.cshl.edu/)with a service charge of $1400 per PCR screen. TUSC (Trait Utility System for Corn) populations are available at no cost from Pioneer Hi-Bred upon signing a collaborative agreement. References 1.) Maize Gene Discovery Project ( MaizeGDB)

Insertional mutagenesis in tomato. Insertion Mutant databases in tomato are comparatively less .Only a few mutant stock databases have data though none pertains to Insertional mutants until now. The Tomato Genetics Resource Centre of Dr CM Ricks at UC Davis, USA The SOL Genomics Network (SGN); Genes that make Tomato . TOMATOMA- A Tomato mutant archive of the University of Tsukuba,Japan

Insertional mutagenesis in tomato has been carried out mostly with the maize Ac/Ds transposable elements. Following the first report showing that Ac/Ds elements are active in tomato, patterns of Ac/Ds transposition in this species have been described. A number of tomato genes have been isolated by transposon tagging with Ac/Ds. These include the Cf-9 and Cf-4 loci , which control resistance to various races of Cladosporium fluvum; Dwarf, a gene encoding a cytochrome p450 homologue defective chloroplasts and leaves (DCL), which controls chloroplast development ; and FEEBLY, a gene involved in metabolism and development .In most cases, these genes were tagged by targeted tagging, that is, by taking advantage of the preferential transposition of Ac/Ds to nearby sites and and of the linkage between the target and previously mapped Ds elements . There has been a concerted effort by European groups to map a large number of Ds elements in the tomato genome to facilitate targeted tagging. Using a system similar to that described previously, the European Union-funded TAGAMAP project has recently completed the mapping of 141 Ds-containing T-DNA inserts spread throughout the 12 chromosomes of tomato (D Gidoni, personal communication). The Defective embryo and meristems (Dem) tomato gene was found in a non-targeted screen for mutant phenotypes in Ac/Ds-tagged plants Despite these successes in transposon tagging in tomato, efficient reverse genetics tools are still needed, especially in light of the continuing release of tomato ESTs into public sequence databases, such as GenBank, and the subsequent assembly of ESTs into tentative genes In tomato, no transposon-tagged population is available yet, although a pilot population of about 3000 lines containing 60009000 insertions has been produced by Emmanuel et al It is estimated that 200000300000 insertion lines, each containing two or three copies of the Ds transposon that inserts preferentially into genes, would be required for nearsaturated mutagenesis in tomato In tomato, the sequencing of insertion sites has been initiated on a small scale using Ds-tagged insertion lines . To date, there are no reports of activation tagging in tomato. References 1.) Emmanuel et al ,Current Opinion in Plant Biology ,Volume 5, Issue 2, 1 April 2002, Pages 112-117.)