REPORTS
Figure 2. SNP genotyping of DNA extracted from individual cotton seeds after 41 PCR cycles. The red rectangle denotes wells that contained DNA samples extracted using a commercial kit and fresh leaf tissue. In the two clusters, wells containing conventionally extracted DNA
samples are denoted by the symbols * and +. Wells containing non-template controls (no sample DNA) are black. All of the other 88 DNA samples were extracted by the non-destructive seed DNA extraction method from a backcross family segregating for the SNP Gl_072641.
ously labeled with a matching unique identi- fying code (Figure 1F). Once coupled, the plates were reverted to upright position and hand-slapped to dislodge and transfer the drillings into the matching wells of the regular PCR plate. The modified plates of sampled seeds were stored sequentially according to their labels. After genotyping, seeds were selected based on genotype, then extracted from the plate and germi- nated in “ragdolls” of germination paper (#SD3815L; Anchor Paper Co., St. Paul, MN) or in Jiffy peat pellets (#JI703; BWI, Schulenburg, TX).
Vol. 58 | No. 5 | 2015
Seedling tissue samples were obtained
from cotyledons at 3–10 days post- emergence (Figure 1G) using a standard (6 mm diameter) single-hole punch (Figure 1H), which was wiped clean between samples. For applications requiring smaller amounts of DNA, a commercially available 3 mm punch or, alternatively, a 200 µl pipette tip can be cut to the appropriate size and used to create 3 mm diameter leaf disks. Each tissue disk was transferred into a well of a 96-well plate or a flat-bottomed microfuge tube, depending on the overall number of samples. Samples were dried
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either by incubating for 1 h in a benchtop oven at 60°C or adding desiccant beads to the wells/tubes, sealing or closing them, and drying for 24 h (desiccant beads were removed before crushing). Once dried, tissues were crushed individually to increase the contact surface. The extraction buffers described below
were added into wells containing seed or dried seedling cotyledon tissue, which were processed according to the DNA extraction procedures for cotton seeds and seedlings listed in Table 1. (See Supplementary Material for detailed protocol.)
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