Plant Reverse Genetics

Volume 678 of the series Methods in Molecular Biology pp 179-190


Reverse Genetics in Medicago truncatula Using Tnt1 Insertion Mutants

  • Xiaofei ChengAffiliated withPlant Biology Division, The Samuel Roberts Noble Foundation
  • , Jiangqi WenAffiliated withPlant Biology Division, The Samuel Roberts Noble Foundation Email author 
  • , Million TadegeAffiliated withPlant Biology Division, The Samuel Roberts Noble Foundation
  • , Pascal RatetAffiliated withInstitut des Sciences du Vegetal, CNRS
  • , Kirankumar S. MysoreAffiliated withPlant Biology Division, The Samuel Roberts Noble Foundation

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Medicago truncatula has been chosen as one of the two model species for legume molecular genetics and functional genomics studies. With the imminent completion of M. truncatula genome sequencing, availability of large-scale mutant populations becomes a priority. Over the last 5 years, nearly 12,000 insertion lines, which represent approximately 300,000 insertions, have been generated at the Samuel Roberts Noble Foundation using the tobacco retrotransposon Tnt1. Individual genomic DNA was isolated from each insertion line and pooled into four levels with the super-pool containing 500 lines. Using Tnt1-specific and gene-specific primers, a PCR-based efficient reverse screening strategy has been developed. Amplified PCR products are purified and sequenced to identify the exact insertion locations. Overall, approximately 90% of genes screened were found to have one or more Tnt1 insertions. Therefore, this PCR-based reverse screening is a rapid way of identifying knock-out mutants for specific genes in Tnt1-tagged population of M. truncatula. In addition to the DNA pool screening, a web-based database with more than 13,000 flanking sequence tags (FSTs) has also been set up. One can search the database to find an insertion line for the gene of interest.

Key words

Medicago truncatula Tnt1 Reverse genetics PCR-based screening Mutants