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Table 1. Procedure for high-throughput DNA extraction from cotton seeds or seedlings 1 2 3
Add 50 µL Buffer A to the samples, then seal the plate. Mix to immerse tissue and then centrifuge briefly to consolidate tissue.
Heat, using one of several options: (1) Microwave for 1 minute at 30% power (approximately 400 Watts); (2) Heat for 10 minutes at 95°C in an oven, a thermocycler, or a waterbath.
4 5 6
Add 50 µL Buffer B to the plate. Mix or vortex, and then centrifuge briefly to consolidate extract and cellular debris.
For cotton seed extract: Dilute 2×, transfer 10 µL to a new plate, then dilute DNA samples 5× or 10× in the new plate.
7 8
For seedling extract: Transfer 10 µL to a new plate, then dilute DNA samples 10× in the new plate. Transfer 2 µL diluted extract to a 96-well FrameStar PCR Plate. Optionally dry the samples at 65°C. Conduct PCR.
completed well before planting. Similarly, DNA extraction and genotyping from very young seedlings would allow MAS prior to transplanting and/or flowering, thereby reducing costs and improving the selection of parents for crossing. Here we report methods for rapid
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extraction of DNA from ungerminated seeds or seedlings suitable for PCR analysis. DNA extraction from single dry seeds of wheat, rice, barley, and several other species has been described previ- ously (13–15), including some patented robotics-enabled methods for tissue sampling (16,17). None of these existing approaches, however, allow DNA extraction from cotton by non-automated, fast, and inexpensive means. Compared to commercial kits suitable for use with cotton seeds and cotyledons, our new methods are a magnitude lower both in cost and time. Being applicable to seeds and seedlings, the new methods enable users to take advantage of genotyping before planting or during the initial seedling stages prior to flowering (e.g., for marker- assisted selection). The efficacy and usefulness of these methods have been verified by their incorporation into several on-going research projects that require low-cost DNA preparations for PCR-based genotyping.
Materials and methods 651-501-2337 Email:
info@qmisystems.com www.qmisystems.com
Plant materials Mature undelinted and delinted seeds were used to assess the applicability of the method to cotton (Gossypium hirsutum) seeds. Cotton seedlings were grown under greenhouse conditions (day temperature: 29.4°C, night temperature: 23.9°C, humidity: 70%, light: natural light) in a commercial growing medium (Metro-Mix 700 or 900; Sun Gro Horticulture, Agawam, MA).
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Cotton seed and seedling tissue sampling procedures and DNA extraction Delinted or undelinted seeds were sanded on the end opposite the funiculus using a sanding drum (Dremel #407; Dremel, Racine, WI) with 60-grit sanding bands (Dremel #408), just far enough to expose cotyledonary tissue (Figure 1A). Using a physically modified PCR plate (#MPS- 499; Phenix Research, Candler, NC) from which the end of each well was previously removed by cutting with a box-cutter, sanded seeds were inserted one seed per well with the sanded end facing the bottom (Figure 1, B and C). Each modified plate was labeled with a unique identi- fying code. Adhesive tape (e.g., packing tape) was applied to the entire plate to prevent the seeds from falling out of the wells. When seeds were variable in size, additional manual pressure was applied as needed to force each seed into a well, and/ or a water-soluble glue (e.g., Helping Hand Carpenter’s glue, #80016; Faucet Queen, Vernon Hills, IL) was applied to the plate to more firmly secure the seeds. The PCR plate was then inverted, and an engraving cutter (Dremel #107) was rotated at a slow speed while inserting it to the depth of the cutting head (~2.4 mm) (Figure 1D) such that the abrasion created fine tissue particles in each well. After each use, the cutting head was cleaned by drilling a hole into a conven- tional pencil eraser (Figure 1E). Approxi- mately 3.5 mg of particulate cotyledonary tissue was obtained from each sample. For applications involving smaller seeds or those requiring smaller amounts of DNA, a 1.6 mm engraving cutter (Dremel #106) was used, which yielded around 1.5 mg of tissue. Once drilling was completed, the still-inverted modified PCR plate was physically placed male-to-female with an inverted non-modified PCR plate previ-
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