Transcript ASA POSTER-2008

Transposon-Mediated Functional Genomics in Barley
Peggy G. Lemaux1, Jaswinder Singh1, Shibo Zhang1, Phil Bregitzer2, Patrick Hayes3, Victoria Carollo4
1Department of Plant and Microbial Biology University of California, Berkeley CA, 94720, 2National Small Grains Germplasm Research Facility, USDA ARS, Aberdeen, ID, 83210, 3Department of Crop and Soil Science, Oregon State University, Corvallis OR, 97331, 4Western Regional Research Center, USDA ARS,
Albany CA, 94710
Specific objectives:
ABSTRACT
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Characterized transposed sites
Nested inverse PCR was used to obtain
5’and 3’ flanking sequences from TNP lines
Transposable elements have advantages over other approaches for determining gene function in large genome cereals. Different
strategies have been used to exploit maize Ac/Ds for such studies in heterologous species. First, large numbers of independent Ds
insertion lines (TNPs) are generated and screened phenotypically. Alternatively, smaller numbers of transposed elements are
identified, mapped and then remobilized for localized gene targeting. A robust platform was developed to use transposon targeting
approaches in barley to complement existing, extensive genomic resources. In our NSF Plant Genome Research Project we (i)
generated 200 single-copy Ds TNPs, (ii) determined flanking sequences in >100 lines, (iii) mapped 43 DsT loci on the genetic
linkage map, (iv) identified 250 BAC addresses using flanking sequence probes and (v) quantified remobilization frequencies of
primary, secondary and tertiary TNPs for saturation mutagenesis schemes. Results from BLAST searches of flanking sequences
indicated that ~86% are from known or putative genes; examples include MLA1, wall-associated kinases, ubiquitin-conjugating
enzyme, ATP-binding transporter, terpene synthase, ankyrin1-like protein and cytochrome P450. Mapped insertion sites are
valuable for tagging linked genes by reactivating Ds elements close to traits/genes of interest to achieve saturation mutagenesis.
Because the marked tendency of Ds to transpose to linked locations is critical for this approach, we investigated characteristics
important to TNP reactivation, i.e., status of terminal inverted repeats ((TIRs) and 8 bp duplications and the nature of insertion
sites. Remobilization frequencies of 12-17% over 3-4 generations of TNPs with intact TIRs provide the necessary foundation for
tagging and “transposon-walking” (repeated localized transposition) strategies. The Bregitzer laboratory, which maintains and
distributes these materials to interested researchers, has continued to generate additional tagging resources. Outreach efforts, which
included the animated educational game, “Genetic Dartboard” (http://barleyworld.org/oregonwolfe.php), were aimed at increasing
understanding of the Ac/Ds transposon tagging system.
Generate Golden Promise lines carrying single, independently transposed Ds-bar elements.
Determine flanking sequence of Ds insertion site in TNP line for bioinformatics analysis.
Map insertion site in OWB, or other mapping populations if necessary.
Generate sufficient transposed lines to achieve one insertion in every bin (5-10 cM).
Assign BAC addresses to flanking sequences.
Develop OWB-D lines with Ds-bar or AcTPase; cross to obtain lines with dispersed Ds
elements and map insertions in OWB DH mapping population.
 Establish repository to provide stocks to scientific and educational community.
 Continue development of OWB educational website as enhanced teaching tool.
Development of primary Ds insertion lines and their activation
Co-transformation of immature embryos of barley with pSP-Ds-Ubi-bar-Ds and
pBS-codA-Act-Ubi-AcTPase (Ac transposase) initiated the primary transpositions.
Transposase- expressing lines were also developed by bombarding immature embryos
of barley with pBS-codA-Act-Ubi-AcTPase or pUC-codA-Act-AcAcTPase separately to
re-activate Ds insertion lines via crossing.
Mapped Ds elements randomly dispersed in the genome
A scheme for activation of Ds element
T1
3’
5’
Ds
Ds
x
Barley line containing transposed Ds
AcTPase
nos
Ubi 1
Act1
Cod A
35S
Line 2
nos
bar
Ubi 1
Primary Transpositions
Line 1
5’
Barley line with active transposase
(32B-1)
Ds transposition
Selection for non AcTPase,
Ds-containing plants
Stable single copy Ds transposants
Ds flanking sequences provide tools to identify
important BAC clones and monitor Ds movement
DNA was digested with EcoRV and probed
with Ds 5’ element (400 bp)
DNA was digested with HindIII and probed
with fragment containing nos+Ds 3’ (650 bp)
Ds transposon insertion lines can be further re-activated by
crossing with transposase-expressing lines
Nos3-1250R
*
BAC libraries provide direct
and reproducible access to the
genome. Utility of BACs is
enhanced by assigning BAC
clones to mapped genomic
regions near genes of interest.
With regard to Ds inserts,
BAC hybridization provides
copy number estimates, that
support gene predictions based
on computer alignments.
1650 bp
1000 bp
850 bp
500 bp
200 bp
*
*
1250R-751F
GP TNP-24 GP TNP-24 GP TNP-24
The TNP lines generated after activation of primary transposants can be further
re-activated via crossing with transposase- expressing lines.
Our goal is to generate a total of 120 Ds insertion lines, scattered throughout the genome.
* *
751F Ds5-379R
*
*
A new F2 population was obtained after crossing
Tnp 24 with pUC-codA-Act-AcAcTPase
Public Webpage to Obtain Materials/Information
~ 12% (19/152) transpositions were obtained
Virtual Barley
Genes for the 2-row vs 6-row phenotype (Vrs1) and for yellow-striped vs. green
leaves (Wst) are tracked through a simple genetic cross using FLASH animation.
An animation to show recombination of these genes during meiosis is in progress.
Ds elements move into genes
DNA was digested with HindIII and probed with Nos+Ds3’ element
* Indicates new transpositoin
Ds
900
650
100
Ds insertion site structure
Active-site ATP-binding
Ds5-142F
Ds5-379R
3,497 BP DS BAR TRANSPOSABLE ELEMENT CASSETTE
Diagnostic primers identify 5’ and 3’ ends of Ds insert.
Nos3
751F
Ds bp 721
1263
70
Calcium-binding
EGF-like domain
Ds3-N
TNP11: Wall-associated protein kinase (722 aa)
Ds
960bp
39F
1.5 kb
1.2 kb
1250R
Barley-mation is educational animation that provides
information on plant anatomy, plant development
and breeding. For example, Flash Animation is used
to show what happens during a simple genetic cross
to genes for the 2-row and 6-row (Vrs1) phenotype
and for yellow-striped and green leaves (Wst).
782bp
1130R
TNP-24 1,450 BP GENOMIC SEQUENCE AND PRIMERS
Primers used for mapping are indicated in red.
Probe used in BAC screening indicated in purple
ABC transporter signature
ATP/GTP-binding
TNP 67: ABC transporter (1182 aa)
TIR –
Terminal Inverted Repeat
Ds 5’ end –
320 bp terminal region from Zea mays Ac transposable element, includes 11 bp TIR
Ds 3’ end –
252 bp terminal region from Zea mays Ac transposable element, includes 11 bp TIR
Ubi promoter – Ubiquitin promoter
Nos terminus – Nopaline synthase 3’ terminus
Bar: coding region for phosphinothricin acetyl transferase (PAT),
providing resistance to glufosinate ammonium herbicide
Exon
Intron
Domain
Ds
A WebPage to serve as a public portal for the mapping results of this project
at wheat.pw.usda.gov/BarleyTNP/IMap displays the Barley bin map with all
mapped TNP’s and previously mapped naked-eye phenotypes (NEPs).
Clicking on a chromosome image from the main page brings up fully
interactive chromosome images. When DsT or NEP loci are clicked, pages
appear that provide extensive information and graphics. Other loci are
linked to their record in the GrainGenes database.
Publications
Cooper, L.D., Marquez-Cedillo, L., Singh, J., Sturbaum, A.K., Zhang, S., Edwards, V., Johnson, K., Kleinhofs, A.,
Rangel, S., Carollo, V., Bregitzer P., Lemaux, P.G. and Hayes, P.M. 2004. Mapping Ds insertions in barley
using a sequence-based approach. Mol. Gen. Genomics DOI: 10.1007/s00438-004-1035-3.
Singh, J., Zhang, S., Lemaux, P. G., Bregitzer, P., Sturbaum, A. K., Edwards, V., Hayes, P. M., Cooper, L. D.,
Marquez-Cedillo, L., Carollo. V. (2005) Maize transposable elements and barley: a new population for
genetic research. Barley Genetics Newsletter 35 :1-2.
Zhang, S., Chen, C., Li, L., Meng, M., Singh, J., Jiang, N., Deng, X-H., He, Z-H., Lemaux, P. G (2005)
Evolutionary expansion, gene Structure, and expression of the rice (Oryza sativa L.) wall-associated kinase
(OsWAKs) gene family. Plant Physiology (In press)
This work is being funded by NSF Award - #0110512