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

doi:10.1111/j.1365-313X.2008.03418.x

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Wiley InterScience

The Plant Journal

Volume 54 Issue 2, Pages 335 - 347

Published Online: 16 Jan 2008

Published in association with the Society for Experimental Biology

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TECHNICAL ADVANCE

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

Million Tadege ¹ , Jiangqi Wen ¹ , Ji He ¹ , Haidi Tu ¹ , Younsig Kwak ^1,† , Alexis Eschstruth ² , Anne Cayrel ² , Gabriella Endre ³ , Patrick X. Zhao ¹ , Mireille Chabaud ⁴ , Pascal Ratet ² and Kirankumar S. Mysore ^1,*

¹ Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA ,
² Institut des Sciences du Vegetale, CNRS, Avenue de la Terrasse, 91198 Gif sur Yvette, France ,
³ Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, Temesvari krt. 62, H-6726 Szeged, Hungary , and
⁴ Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, INRA-CNRS, BP27, F-31326 Castanet-Tolosan Cedex, France

Correspondence to ^*(fax +1 580 224 6692; e-mail ksmysore@noble.org).

^† Present address: Department of Plant Pathology, 367A Johnson Hall, PO Box 646430, Washington State University, Pullman, WA 99164 6430, USA.

KEYWORDS

Tnt1 tagging • Medicago • legume • retrotransposon • insertion mutagenesis • functional genomics

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.