Development and Application of Medicago truncatula Mutant Resources

Medicago truncatula is a model species for legume genetics, genomics, and functional genomics studies. It has been utilized for research in many areas, including plant nutrition, metabolism, growth and development, and plant-microbial and plant-environmental interactions. To better facilitate deciphering gene functions in M. truncatula and other legume species, we have generated more than 21,000 Tnt1 retrotransposon insertion lines, encompassing approximately 500,000 insertions in the genome of M. truncatula, at The Samuel Roberts Noble Foundation. Two reverse genetics platforms have being established: one is a searchable web-based database, which currently contains more than 30,000 Tnt1-flanking sequence tags (FSTs) mapped to pseudo-chromosomes (http://bioinfo4.noble.org/mutant/); the other is a PCR-based reverse screening, which provides screening service for genes-of-interest in genomic DNA pools of insertion lines. Over the past five years, approximately 900 genes have been screened by the PCR approach. One or more Tnt1 insertions have been successfully identified in 85% of these genes, ranging from ~0.5 to 22 kb with genomic or cDNA sequences. So far, we have distributed more than 3600 Tnt1 lines to scientists in 24 countries. To testify the utilization of the reverse genetics resources, two floral homeotic MADS-box genes, MtPISTILATA (MtPI) and MtAGAMOUS (MtAG), were screened for mutant lines in the Tnt1 DNA pools and were searched for insertion lines in the FST database. Two Tnt1 insertion lines were obtained for each gene. Molecular and genetic analyses indicate that Tnt1 insertions in exons of the genes are responsible for the defective floral development in both mtpi and mtag mutants.