* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download ASA POSTER-2008
Pharmacogenomics wikipedia , lookup
Primary transcript wikipedia , lookup
Point mutation wikipedia , lookup
Oncogenomics wikipedia , lookup
Human genetic variation wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Gene desert wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Metagenomics wikipedia , lookup
Ridge (biology) wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Biology and consumer behaviour wikipedia , lookup
Gene expression profiling wikipedia , lookup
Gene expression programming wikipedia , lookup
Genetic engineering wikipedia , lookup
Short interspersed nuclear elements (SINEs) wikipedia , lookup
Genomic imprinting wikipedia , lookup
Human genome wikipedia , lookup
Quantitative trait locus wikipedia , lookup
Non-coding DNA wikipedia , lookup
Pathogenomics wikipedia , lookup
Public health genomics wikipedia , lookup
Minimal genome wikipedia , lookup
Genomic library wikipedia , lookup
Designer baby wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
History of genetic engineering wikipedia , lookup
Microevolution wikipedia , lookup
Genome (book) wikipedia , lookup
Transposable element wikipedia , lookup
Genome editing wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
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 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