Insertional mutagenesis of Brachypodium distachyon using the Tnt1 retrotransposable element

Abstract

SUMMARY Brachypodium distachyon is an annual C3 grass used as a monocot model system in functional genomics research. Insertional mutagenesis is a powerful tool for both forward and reverse genetics studies. In this study, we explored the possibility of using the tobacco retrotransposon Tnt1 to create a transposon‐based insertion mutant population in B. distachyon. We developed transgenic B. distachyon plants expressing Tnt1 (R0) and in the subsequent regenerants (R1) we observed that Tnt1 actively transposed during somatic embryogenesis, generating an average of 6.37 insertions per line in a population of 19 independent R1 regenerant plants analyzed. In seed‐derived progeny of R1 plants, Tnt1 segregated in a Mendelian ratio of 3:1 and no new Tnt1 transposition was observed. A total of 126 flanking sequence tags (FSTs) were recovered from the analyzed R0 and R1 lines. Analysis of the FSTs showed a uniform pattern of insertion in all the chromosomes (1–5) without any preference for a particular chromosome region. Considering the average length of a gene transcript to be 3.37 kb, we estimated that 29 613 lines are required to achieve a 90% possibility of tagging a given gene in the B. distachyon genome using the Tnt1‐based mutagenesis approach. Our results show the possibility of using Tnt1 to achieve near‐saturation mutagenesis in B. distachyon, which will aid in functional genomics studies of other C3 grasses.

Medicago truncatula resources to study legume biology and symbiotic nitrogen fixation

“…With a 10-year effort, we have generated a large collection of Tnt1 insertion lines (∼21,000) in M. truncatula that nearly saturate the genome with more than 1,000,000 Tnt1 insertions [ 2 , 30 , 58 ]. Tnt1 -based insertional mutagenesis was applied in many plant species including L. japonicus [59] , M. truncatula [30] , Brachypodium distachyon [60] , lettuce [61] , soybean [62] and potato [63] . The legume research community greatly benefitted from the use of M. truncatula Tnt1 -insertion population and made important discoveries by dissecting the pathways of symbiotic nitrogen fixation, root nodulation, secondary metabolites, inflorescence and floral architecture, pollen development, seed development, and other aspects of legume biology [ 25 , 26 , [64] , [65] , [66] , [67] , [68] , [69] , [70] , [71] , [72] , [73] , [74] , [75] , [76] ].…”

“…The more recent next generation sequencing technologies aimed to sequence the FST regions directly through the use of whole genome sequencing (WGS) was demonstrated [78] . Recent methodology of FST identification using WGS demonstrated the identification and presence of substantially more Tnt1 insertions in the mutant genomes [ 60 , 78 ]. Seqcap, developed on the groundwork of exome capture, is more economical and faster than WGS.…”

“…Seqcap, developed on the groundwork of exome capture, is more economical and faster than WGS. Seqcap was able to recover most of the Tnt1 insertions in a given line and was comparable to WGS methodologies [ 2 , 60 ].…”

Tracing Mobile DNAs: From Molecular to Population Scales

“…For example, Tos17 in rice is specifically activated by in vitro tissue culture and the resulting random insertional mutants tagged with Tos17 are important genetic resources in the rice functional genomics ( Hirochika et al, 1996 ; Hirochika, 2010 ). Similarly, Tnt1 was used to generate genetic mutants in Medicago truncatula , Brachypodium distachyon , and Glycine max ( D’Erfurth et al, 2003 ; Tadege et al, 2008 ; Revalska et al, 2011 ; Cui et al, 2012 ; Nandety et al, 2020 ), and the maize Ac/Ds DNA transposon system was used as a functional genomics tool in Arabidopsis , Oryza sativa , and Glycine max ( Long et al, 1993 ; Mathieu et al, 2009 ; Wang et al, 2013 ). Despite the vast importance of transposons, little is known about the regulatory mechanisms of their mobilization, which is largely because of the lack of experimental methods that can detect the transposition events with sufficient sensitivity and precision.…”

Construction of a Full-Length cDNA Over-Expressing Library to Identify Valuable Genes from Populus tomentosa

“…In recent decades, loss-of-function mutant libraries constructed using T-DNA or transposon insertional mutagenesis approaches have been widely used in many plant species [ 5 , 6 , 7 , 8 , 9 ]. Numerous novel genes have been successfully cloned and characterized.…”