Figure 1.
Forward Genetic Screen to Identify Genes Involved in the Maintenance of Organelle Genome Topology.
Schematic representation of the different steps of the forward genetic screen. Plants with white first true leaves represent the mutants sensitive to ciprofloxacin (CIP) or novobiocin (NOVO).
Figure 2.
Overview of Targeted Genomic Sequencing.
Blue rectangles represent genomic DNA, and red rectangles correspond to T-DNA insertions. The grey squares represent the 454 specific primers added in order to bind the sequencing beads (purple circles). The green circles correspond to biotin bound to a red T-DNA specific primer and hybridized to T-DNA. Hybridized sequences are then enriched by capture on streptavidin beads (orange circles).
Figure 3.
Coverage of the pSKI015 Vector Obtained by Sequencing.
Features of the pSKI015 are summarized below the coverage graph. The blue rectangles represent the T-DNA cassette with the right (RB) and left (LB) borders in green. The position of the 35 S enhancers are indicated by blue open end arrows. The red lines represent the annealing regions of the three biotinylated primers for each border. The position where the repeated reads align is indicated by the double red arrowhead line on the coverage graph.
Figure 4.
Association of an Insertion Event to a Specific Line by 2D-PCR Pooling.
A. Workflow of the 2D-PCR pooling B. An example of the pooling design for 16 plants. Each plant genomic DNA is pooled in a unique set combination. The plants encompass by the colored rectangle associate to the pool of the same color. C. Data analysis to identify the positive line. All bands on a given gel correspond to the same amplification product in different pools.
Figure 5.
Schematic Illustration of the Insertion Sites in the Three Novobiocin-Sensitive Mutant Lines.
The small black arrows represent the orientation of the CaMV 35S enhancers within the T-DNA (rectangle). For Insertion 3, a different part of the plasmid still containing the enhancer region has been inserted.