Rice Genetics IV - IRRI books - International Rice Research Institute

Transposon library generation10 starter enhancer trap linesT 12,000 total seeds (200 seeds each)Selection for BAR heterozygotes (1/2)1,000 selfed plants selectedSU1BART 2200,000 total seedsSelection for BAR heterozygotes (1/2)100,000 selfed plants selectedSU1BART 320,000,000 total seedsBAR-SU1 selectionSU1 negative (1/4) = 5,000,000{ }Frequency of independent transpositions (10%) = 500,000Frequency of unlinked transpositions (30%)150,000 stable independent insertionsSU1SU1BARBARFig. 5. Transposon library generation. Using a minimum of 10 active single-locus starter enhancertrap lines, with a multiplication rate of 200 seeds per plant (Nipponbare), a total of200,000 T 2 seeds can be obtained from heterozygous plants that are identified on the basis ofprogeny-test segregation for Basta R . Similarly, from the 200,000 plants, half are heterozygotesand are identified by progeny tests in the T 3 generation, in which 20 million seeds areproduced from the heterozygous parents and can be used for selection of the population ofstable transposants. A sketch of the T-DNA carrying the SU1 and BAR (on transposon) markersis shown alongside. Using Basta R and SU1 selection, only the plants bearing a Basta Rtransposed element (BAR + SU1 – ) are selected, whereas all SU1-bearing plants (withtransposase) are rejected. The frequency of independent transposition was observed to be10% and that of unlinked transposition about 30%, which will generate about 150,000 stableindependent insertions.274 Greco et al