The flowers of major cereals are arranged on reproductive branches known as spikelets, which group together to form an inflorescence. Diversity for inflorescence architecture has been exploited ...during domestication to increase crop yields, and genetic variation for this trait has potential to further boost grain production. Multiple genes that regulate inflorescence architecture have been identified by studying alleles that modify gene activity or dosage; however, little is known in wheat. Here, we show TEOSINTE BRANCHED1 (TB1) regulates inflorescence architecture in bread wheat (Triticum aestivum) by investigating lines that display a form of inflorescence branching known as “paired spikelets.” We show that TB1 interacts with FLOWERING LOCUS T1 and that increased dosage of TB1 alters inflorescence architecture and growth rate in a process that includes reduced expression of meristem identity genes, with allelic diversity for TB1 found to associate genetically with paired spikelet development in modern cultivars. We propose TB1 coordinates formation of axillary spikelets during the vegetative to floral transition and that alleles known to modify dosage or function of TB1 could help increase wheat yields.
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Using a two-element iAc/Ds transposon-tagging system, we identified a rice (Oryza sativa L. cv Nipponbare) recessive mutant, anther indehiscence1 (aid1), showing partial to complete spikelet ...sterility. Spikelets of the aid1 mutant could be classified into three types based on the viability of pollen grains and the extent of anther dehiscence. Type 1 spikelets (approximately 25%) were sterile due to a failure in accumulation of starch in pollen grains. Type 2 spikelets (approximately 55%) had viable pollen grains, but anthers failed to dehisce and/or synchronize with anthesis due to failure in septum degradation and stomium breakage, resulting in sterility. Type 3 spikelets (approximately 20%) had normal fertility. In addition, aid1 mutant plants had fewer tillers and flowered 10 to 15 d later than the wild type. The Ds insertion responsible for the aid1 mutation was mapped within the coding region of the AID1 gene on chromosome 6, which is predicted to encode a novel protein of 426 amino acids with a single MYB domain. The MYB domain of AID1 is closely related to that of the telomere-binding proteins of human, mouse, and Arabidopsis, and of single MYB domain transcriptional regulators in plants such as PcMYB1 and ZmIBP1. AID1 was expressed in both the leaves and panicles of wild-type plants, but not in mutant plants.
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We evaluated a two-component transposon iAc/Ds system for generating a library of insertional mutants in rice. The constructs used have gene or enhancer trapping properties, plasmid rescue and ...T-DNA/Ds launching pad reporter facilities. Mutagenic iAc/Ds lines were produced by three methods: crossing iAc and Ds containing lines; co-transformation with iAc and Ds constructs; and super-transformation of iAc transgenic calli with Ds constructs. First and second generation screening populations, derived from crosses (F2 and F3) or double transformation (DtT1 and DtT2), were analysed for stable insertion lines containing Ds transposed to locations unlinked to iAc. The average frequencies of putative stable insertion (PSI) lines in the F2, DtT1, F3 and DtT2 populations were 6.61, 5.58, 11.47 and 7.05% respectively, with large variations in these frequencies in screening populations derived from different mutagenic lines. Further analyses indicated that 41, 33, 65 and 64% of the PSI lines, respectively, have Ds transposed to locations unlinked to the original Ds launching pad. Using the plasmid rescue system, sequences flanking Ds from 137 PSI lines were obtained. Sixty-eight of these lines had unique insertions in genomic regions, of which 18 were known sequences. Because the average frequency of proven stable insertion lines in any of our screening populations has been less than 5%, we suggest that additional features should be incorporated in this two-component iAc/Ds system to increase the screening efficiency, and to make it suitable for large-scale insertional mutagenesis and determination of gene function in rice.
To gain further understanding of the mechanisms involved in Agrobacterium-mediated genetic transformation and T-DNA integration, we analysed 156 T-DNA/rice, 69 T-DNA/T-DNA and 11 T-DNA/vector ...backbone (VB) junctions, which included 171 left borders (LB) and 134 right borders (RB). Conserved cleavage was observed in 6% of the LB and 43% of the RB. Terminal microhomology (1–10bp) was identified in 58% of T-DNA/rice, 43% of T-DNA/T-DNA and 82% of T-DNA/VB junctions, and this occurred particularly at the LB junctions. Approximately 32% of both T-DNA/rice and T-DNA/T-DNA junctions harboured 1–344bp of filler DNA that was derived mainly from the T-DNA region adjacent to the breakpoint and/or from the rice genome flanking the T-DNA integration site. Structure of the filler DNA was more complicated at the T-DNA/T-DNA junction than at the T-DNA/rice junction, indicating the presence of T-DNA recombination or rearrangement prior to or during T-DNA integration. When two T-DNAs were integrated in the inverted repeat configuration, significant truncation was always observed in one of the two T-DNAs whereas with direct repeat configuration, a large truncation was less frequent. Most integration events analysed in this study could be addressed by previously proposed models; however, the characteristics of the T-DNA repeats and the complicated filler DNA between two T-DNA copies imply that multiple mechanisms are involved in the formation of T-DNA repeats as well as in T-DNA integration in plants.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
We have developed a transiently-expressed transposase (TET)-mediated Dissociation (Ds) insertional mutagenesis system for generating stable insertion lines in rice which will allow localized ...mutagenesis of a chromosomal region. In this system, a Ds containing T-DNA construct was used to produce Ds launch pad lines. Callus tissues, from single-copy Ds/T-DNA lines, were then transiently infected with Agrobacterium harbouring an immobile Ac (iAc) construct, also containing a green fluorescent protein gene (sgfpS65T) as the visual marker. We have regenerated stable Ds insertion lines at a frequency of 9-13% using selection for Ds excision and GFP counter selection against iAc and nearly half of them were unique insertion lines. Double transformants (iAc/Ds) were also obtained and their progeny yielded approximately 10% stable insertion lines following excision and visual marker screening with 50% redundancy. In general, more than 50% of the Ds reinsertions were within 1 cM of the launch pad. We have produced a large number of single-copy Ds/T-DNA launch pads distributed over the rice chromosomes and have further refined the Ds/T-DNA construct to enrich for “clean” single-copy T-DNA insertions. The availability of single copy “clean” Ds/T-DNA launch pads will facilitate chromosomal region-directed insertion mutagenesis. This system provides an opportunity for distribution of gene tagging tasks among collaborating laboratories on the basis of chromosomal locations.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
A robust Laboratory Information Management System (LIMS) is required for the efficient handling of data generated from large-scale insertional mutagenesis projects. The Rice Gene Machine Information ...Management System (RGMIMS), a web-based modular LIMS, developed in a rice functional genomics laboratory at CSIRO, currently has four core modules: Seed Management, Transformation Management, Plant/Progeny Management, Phenotype Management, and an ad hoc querying module. RGMIMS manages, preserves and tracks large inventories of transgenic germplasm and enables efficient and accurate record keeping of the large quantities of experimental data. RGMIMS automates and seamlessly integrates multi-step experimental processes. A web user interface, incorporating barcoding utilities, enables rapid data capture and tracking of biological resources. Ontologies from Gramene and Plant Ontology consortium are used to describe mutant phenotypes. RGMIMS supports generic research processes in plant mutagenesis and could readily be adapted to general LIMS for high-throughput plant research.
Using a two-element iAc/Ds transposon-tagging system, we identified a rice (Oryza sativa L. cv Nipponbare) recessive mutant, anther indehiscence1 (aid1), showing partial to complete spikelet ...sterility. Spikelets of the aid1 mutant could be classified into three types based on the viability of pollen grains and the extent of anther dehiscence. Type 1 spikelets (approximately 25%) were sterile due to a failure in accumulation of starch in pollen grains. Type 2 spikelets (approximately 55%) had viable pollen grains, but anthers failed to dehisce and/or synchronize with anthesis due to failure in septum degradation and stomium breakage, resulting in sterility. Type 3 spikelets (approximately 20%) had normal fertility. In addition, aid1 mutant plants had fewer tillers and flowered 10 to 15 d later than the wild type. The Ds insertion responsible for the aid1 mutation was mapped within the coding region of the AID1 gene on chromosome 6, which is predicted to encode a novel protein of 426 amino acids with a single MYB domain. The MYB domain of AID1 is closely related to that of the telomere-binding proteins of human, mouse, and Arabidopsis, and of single MYB domain transcriptional regulators in plants such as PcMYB1 and ZmIBP1. AID1 was expressed in both the leaves and panicles of wild-type plants, but not in mutant plants.
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A conditional negative selection marker is essential for high throughput insertional mutagenesis with any two-element transposon tagging system. Thetms2 gene encodes indoleacetic acid hydrolase ...(IAAH) which converts naphthaleneacetamide (NAM) to the potent auxin naphthaleneacetic acid, a phytotoxic derivative. This gene, under the control of the manopine synthase gene 2 promoter fromAgrobacterium tumefaciens and exogenously applied NAM, have been used effectively as a negative selector inAc/Ds insertional mutagenesis ofArabidopsis thaliana (Sundaresan et al., 1995). In this study we show thattms2 can also be used as a negative selector in rice. T^sub 1^ transgenic seedlings expressing thistms2 gene under the control of themas2' promoter showed significant reduction in shoot and root growth in the presence of 5-10 μM NAM under specified growth conditions compared to plants not containing this gene.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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