An Efficient System for Ds Transposon Tagging in Brachypodium distachyon

Abstract

Transposon tagging is a powerful tool that has been widely applied in several species for insertional mutagenesis in plants. Several efforts have aimed to create transfer-DNA (T-DNA) insertional mutant populations in Brachypodium distachyon, a monocot plant used as a model system to study temperate cereals, but there has been a lack of research aimed at using transposon strategies. Here, we describe the application of a maize (Zea mays) Dissociation (Ds) transposon tagging system in B. distachyon. The 35S::AcTPase cassette and Ds element were constructed within the same T-DNA and transformed into B. distachyon plants. The Ds element was readily transposed to other chromosomes or to the same chromosome under the function of Activator (Ac) transposase. Through homologous chromosome synapsis, recombination, and segregation, the Ds element separated from the Ac element. We selected stable Ds-only plants using G418 and GFP assays and analyzed 241 T 0 lines, some of which were highly efficient at producing Ds-only progeny. Through thermal asymmetric interlaced PCR, we isolated 710 independent Ds flanking sequences from Ds-only plants. Furthermore, we identified a large collection of mutants with visible developmental phenotypes via this transposon tagging system. The system is relatively simple and rapid in comparison to traditional T-DNA insertion strategies, because once efficiency lines are obtained they can be reused to generate more lines from nontransposed plants without the use of time-consuming tissue culture steps.

Insertional mutagenesis of Brachypodium distachyon using the Tnt1 retrotransposable element

“…Insertional mutagenesis has the added advantage that it identifies the mutated genes by forward genetics following the mutant phenotypes, unlike mutants generated by chemicals (using ethyl methane sulfonate) and ionizing radiation (using fast neutrons, c-rays and X-rays) (Tadege et al, 2005). Different approaches have been taken to generate mutants in B. distachyon (Vain et al, 2008;Dalmais et al, 2013), along with transposon tagging (Wu et al, 2019). Currently, there is only a limited availability of cloning friendly mutants in B. distachyon.…”

“…Thus, DNA transposons (class 2) such as maize Ac/Ds elements have been popularly used in plant species such as tomato (Carter et al, 2013), Arabidopsis (Long et al, 1993) and, more recently, in B. distachyon (Wu et al, 2019). The maize Disassociation (Ds) transposon tagging system was used to screen 241 B. distachyon T 0 lines that resulted in the identification of 710 independent Ds flanking sequences (Wu et al, 2019). On the other hand, class 1 elements that use a RNA intermediate are divided into non-long terminal repeat (LTR) retrotransposons and LTR retrotransposons.…”

Cloning of Maize TED Transposon into Escherichia coli Reveals the Polychromatic Sequence Landscape of Refractorily Propagated Plasmids

“…However, the use of Mu elements may be short of achieving genome saturation, largely because of the insertion preferences evident from the sequence analysis of independently constructed MuDR/Mu insertional libraries [ 39 ]. In addition, the application of the MuDR/Mu system to the production of plant mutant collections is still confined to maize in contrast to the maize Ac / Ds system, which has been deployed in several plant species [ 40 , 41 , 42 , 43 , 44 , 45 , 46 ].…”