Reverse Genetics in Medicago truncatula Using Tnt1 Insertion Mutants
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Last Update: 2020-11-19
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Source: Internet
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Author: User
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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.
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