Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula

Million Tadege, Jiangqi Wen, Ji He, Haidi Tu, Younsig Kwak, Alexis Eschstruth, Anne Cayrel, G. Endré, Patrick X. Zhao, Mireille Chabaud, Pascal Ratet, Kirankumar S. Mysore

Research output: Contribution to journalArticle

251 Citations (Scopus)

Abstract

Medicago truncatula is a fast-emerging model for the study of legume functional biology. We used the tobacco retrotransposon Tnt1 to tag the Medicago genome and generated over 7600 independent lines representing an estimated 190 000 insertion events. Tnt1 inserted on average at 25 different locations per genome during tissue culture, and insertions were stable during subsequent generations in soil. Analysis of 2461 Tnt1 flanking sequence tags (FSTs) revealed that Tnt1 appears to prefer gene-rich regions. The proportion of Tnt1 insertion in coding sequences was 34.1%, compared to the expected 15.9% if random insertions were to occur. However, Tnt1 showed neither unique target site specificity nor strong insertion hot spots, although some genes were more frequently tagged than others. Forward-genetic screening of 3237 R1 lines resulted in identification of visible mutant phenotypes in approximately 30% of the regenerated lines. Tagging efficiency appears to be high, as all of the 20 mutants examined so far were found to be tagged. Taking the properties of Tnt1 into account and assuming 1.7 kb for the average M. truncatula gene size, we estimate that approximately 14 000-16 000 lines would be sufficient for 90% gene tagging coverage in M. truncatula. This is in contrast to more than 500 000 lines required to achieve the same saturation level using T-DNA tagging. Our data demonstrate that Tnt1 is an efficient insertional mutagen in M. truncatula, and could be a primary choice for other plant species with large genomes.

Original languageEnglish
Pages (from-to)335-347
Number of pages13
JournalPlant Journal
Volume54
Issue number2
DOIs
Publication statusPublished - Apr 2008

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Medicago truncatula
insertional mutagenesis
Retroelements
Insertional Mutagenesis
retrotransposons
Fabaceae
legumes
Genome
Genes
genome
genes
Medicago
mutants
Mutagens
Genetic Testing
Tobacco
tissue culture
tobacco
Soil
screening

Keywords

  • Functional genomics
  • Insertion mutagenesis
  • Legume
  • Medicago
  • Retrotransposon
  • Tnt1 tagging

ASJC Scopus subject areas

  • Plant Science

Cite this

Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula. / Tadege, Million; Wen, Jiangqi; He, Ji; Tu, Haidi; Kwak, Younsig; Eschstruth, Alexis; Cayrel, Anne; Endré, G.; Zhao, Patrick X.; Chabaud, Mireille; Ratet, Pascal; Mysore, Kirankumar S.

In: Plant Journal, Vol. 54, No. 2, 04.2008, p. 335-347.

Research output: Contribution to journalArticle

Tadege, M, Wen, J, He, J, Tu, H, Kwak, Y, Eschstruth, A, Cayrel, A, Endré, G, Zhao, PX, Chabaud, M, Ratet, P & Mysore, KS 2008, 'Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula', Plant Journal, vol. 54, no. 2, pp. 335-347. https://doi.org/10.1111/j.1365-313X.2008.03418.x
Tadege, Million ; Wen, Jiangqi ; He, Ji ; Tu, Haidi ; Kwak, Younsig ; Eschstruth, Alexis ; Cayrel, Anne ; Endré, G. ; Zhao, Patrick X. ; Chabaud, Mireille ; Ratet, Pascal ; Mysore, Kirankumar S. / Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula. In: Plant Journal. 2008 ; Vol. 54, No. 2. pp. 335-347.
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AU - Cayrel, Anne

AU - Endré, G.

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