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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., Significance Statement Retrotransposable elements transpose from one position in the DNA to another during embryogenesis, causing mutations at the genomic landing site. Here we show that Tnt1, a retrotransposable element from tobacco, can transpose in a monocot plant, Brachypodium distachyon. Based on the analyses of Tnt1 insertions in B. distachyon, we estimated that 29 613 lines would be needed to achieve a 90% probability of tagging every gene in the B. distachyon genome.

Most major crops are polyploid species and the production of genetically engineered cultivars normally requires the introgression of transgenic or gene-edited traits into elite germplasm. Thus, a main goal of plant research is the search of systems to identify dominant mutations. In this article, we show that the Tnt1 element can be used to identify dominant mutations in allogamous tetraploid cultivated alfalfa. Specifically, we show that a single allelic mutation in the MsNAC39 gene produces multifoliate leaves (mfl) alfalfa plants, a pivot trait of breeding programs of this forage species. Finally, we discuss the potential application of a combination of preliminary screening of beneficial dominant mutants using Tnt1 mutant libraries and genome editing via the CRISPR/Cas9 system to identify target genes and to rapidly improve both autogamous and allogamous polyploid crops. Instituto de Biotecnología Fil: Jozefkowicz, Cintia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Jozefkowicz, Cintia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Jozefkowicz, Cintia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina Fil: Gomez, Maria Cristina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Gomez, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gomez, Maria Cristina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina Fil: Odorizzi, Ariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Manfredi; Argentina Fil: Iantcheva, Anelia. Agricultural Academy. AgroBioInstitute; Bulgaria Fil: Ratet, Pascal. Centre National de la Recherche Scientifique (CNRS). Institut National de Recherche pour l’agriculture, l’alimentation et l’environnement (INRAE). Université Paris-Saclay; Francia Fil: Ratet, Pascal. Institute of Plant Sciences Paris-Saclay. Université d’Évry; Francia Fil: Ayub, Nicolás Daniel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Ayub, Nicolás Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ayub, Nicolás Daniel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina Fil: Soto, Gabriela Cinthia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Soto, Gabriela Cinthia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Soto, Gabriela Cinthia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética; Argentina

Most major crops are polyploid species and the production of genetically engineered cultivars normally requires the introgression of transgenic or gene-edited traits into elite germplasm. Thus, a main goal of plant research is the search of systems to identify dominant mutations. In this article, we show that the Tnt1 element can be used to identify dominant mutations in allogamous tetraploid cultivated alfalfa. Specifically, we show that a single allelic mutation in the MsNAC39 gene produces multifoliate leaves (mfl) alfalfa plants, a pivot trait of breeding programs of this forage species. Finally, we discuss the potential application of a combination of preliminary screening of beneficial dominant mutants using Tnt1 mutant libraries and genome editing via the CRISPR/Cas9 system to identify target genes and to rapidly improve both autogamous and allogamous polyploid crops.

Because of its highly efficient homologous recombination, the moss Physcomitrella patens is a model organism particularly suited for reverse genetics, but this inherent characteristic limits forward genetic approaches. Here, we show that the tobacco (Nicotiana tabacum) retrotransposon Tnt1 efficiently transposes in P. patens, being the first retrotransposon from a vascular plant reported to transpose in a bryophyte. Tnt1 has a remarkable preference for insertion into genic regions, which makes it particularly suited for gene mutation. In order to stabilize Tnt1 insertions and make it easier to select for insertional mutants, we have developed a two-component system where a mini-Tnt1 with a retrotransposition selectable marker can only transpose when Tnt1 proteins are co-expressed from a separate expression unit. We present a new tool with which to produce insertional mutants in P. patens in a rapid and straightforward manner that complements the existing molecular and genetic toolkit for this model species., Work done at CRAG was supported by the Ministerio de Ciencia y Innovación (grants: BFU2009-11932 and AGL2013-43244-R). We thank Mitsuyasu Hasebe (National Institute for Basic Biology, Okazaki, Japan) for providing the p35S-loxP-Zeo vector.

Tnt1 (transposable element if Nicotiana tabaccum cell type 1) is one of the very few active LTR retrotransposons used for gene tagging in plants. In the model legume Medicago truncatula, Tnt1 has been effectively used as a gene knock-out tool to generate several very useful mutants. stenofolia (stf) is such a mutant identified by Tnt1 insertion in a WUSCHEL-like homeobox transcription factor. STF is required for blade outgrowth, leaf vascular patterning and female reproductive organ development in barrel medic and woodland tobacco. Using transcript profiling and metabolite analysis, we uncovered that mutant leaves are compromised in steady-state levels of multiple phytohormones, sugar metabolites and derivatives including flavonoids and polyamines. In the lam1 mutant (caused by deletion of the STF ortholog in Nicotiana sylvestris), while glucose, fructose, mannose, galactose, myo-inositol and aromatic aminoacids are dramatically reduced, sucrose is comparable to wild-type levels, and glutamine, proline, putrescine, nicotine and sorbitol are highly increased. We demonstrated that both stf and lam1 mutants accumulate reduced levels of free auxin and ABA in their leaves, and ectopic expression of STF in tobacco leads to auxin and cytokinin overproduction phenotypes including formation of tumors on the roots and crown. These data suggest that STF mediated integration of metabolic and hormonal signals are required for lateral organ morphogenesis and elaboration.

Background Transposable elements constitute an important part of the genome and are essential in adaptive mechanisms. Transposition events associated with phenotypic changes occur naturally or are induced in insertional mutant populations. Transposon mutagenesis results in multiple random insertions and recovery of most/all the insertions is critical for forward genetics study. Using genome next-generation sequencing data and appropriate bioinformatics tool, it is plausible to accurately identify transposon insertion sites, which could provide candidate causal mutations for desired phenotypes for further functional validation. Results We developed a novel bioinformatics tool, ITIS (Identification of Transposon Insertion Sites), for localizing transposon insertion sites within a genome. It takes next-generation genome re-sequencing data (NGS data), transposon sequence, and reference genome sequence as input, and generates a list of highly reliable candidate insertion sites as well as zygosity information of each insertion. Using a simulated dataset and a case study based on an insertional mutant line from Medicago truncatula, we showed that ITIS performed better in terms of sensitivity and specificity than other similar algorithms such as RelocaTE, RetroSeq, TEMP and TIF. With the case study data, we demonstrated the efficiency of ITIS by validating the presence and zygosity of predicted insertion sites of the Tnt1 transposon within a complex plant system, M. truncatula. Conclusion This study showed that ITIS is a robust and powerful tool for forward genetic studies in identifying transposable element insertions causing phenotypes. ITIS is suitable in various systems such as cell culture, bacteria, yeast, insect, mammal and plant. Electronic supplementary material The online version of this article (doi:10.1186/s12859-015-0507-2) contains supplementary material, which is available to authorized users.

Allopolyploidy is a major driving force in plant evolution and can induce rapid structural changes in the hybrid genome. As major components of plant genomes, transposable elements are involved in these changes. In a previous work, we observed turnover of retrotransposon insertions in natural allotretraploid tobacco (Nicotiana tabacum). Here, we studied the early stages of allopolyploid formation by monitoring changes at retrotransposon insertion sites in the Th37 synthetic tobacco. • We used sequence-specific amplification polymorphism (SSAP) to study insertion patterns of two populations of the Tnt1 retrotransposon in Th37 S4 generation plants, and characterized the nature of polymorphic insertion sites. • We observed significant amplification of young Tnt1 populations. Newly transposed copies were amplified from maternal elements and were highly similar to Tnt1A tobacco copies amplified in response to microbial factors. A high proportion of paternal SSAP bands were not transmitted to the hybrid, corresponding to various rearrangements at paternal insertion sites, including indels or the complete loss of the Tnt1/flanking junction. • These data indicate that major changes, such as retrotransposon amplification and molecular restructuring in or around insertion sites, occur rapidly in response to allopolyploidy.

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