REPORTS
tions for a marker that is heterozygous in the F1 hybrid parent and homozygous in the other parent. Each cotyledon-based data point fell along the same ray (angle) as the respective control samples that had the same genotype. The observed ratio in this batch of 36 BC2F1 seedlings was 19:17, which does not differ significantly from the expected testcross ratio (1:1). Relative to conventionally prepared DNAs from segre- gating plants, the experimentally prepared seedling samples were well-clustered along the same rays (angles) as the respective controls, indicating similar SNP-specific fluorochrome ratios. They were also well- clustered in terms of their distance from the origin, indicating similar variation in overall fluorescence amplitude. We also observed similar levels of dispersion at right angles to the ray, indicating that SNP-specific fluoro- chrome ratios were similar for the cotyledon- based extractions and conventional leaf- based extractions. The cotyledon-based extractions were less variable in signal amplitude than the non-destructive seed- based extractions. The cotyledon-extracted DNA samples also needed a few more PCR cycles than the conventional leaf-extracted DNA samples. The procedures for seedling cotyledon
DNA extraction were initially tested on fresh seedling tissue, but the results were inconsistent and generally undesirable (data not shown). Given the relatively high water content and low nuclear density of fresh seedling cotyledon tissue versus seed cotyledon tissue, we tested two facile methods for drying the seedling tissue and then crushed the dried tissue to increase the surface area before adding the buffers. SNP amplification rates were higher for DNA samples extracted from 60 samples dried with desiccant beads (100%) than 136 oven-dried samples (87.5%)., but the latter consumed less time and labor. Drying with lyophilizers would presumably work well, too, but they are not available in many labora- tories. It would be highly advantageous to
breeders and researchers if they could use marker-based selection to choose which seeds to germinate. We initially tested whether or not seeds would germinate after DNA extraction by drilling slightly into 100 seeds, and then tested germination ability using conventional in-lab germination proce- dures. All but 2 of the 100 seeds germinated (Figure 4A), and upon inspection we noted that those 2 seeds had been drilled in the
Vol. 58 | No. 5 | 2015
wrong position (near the radical). Overall, germination was excellent in laboratory and greenhouse conditions; seedlings were vigorous, and no ill effects have been observed, except for the absence of marginal tissue (Figure 4B). In subsequent ragdoll germination tests, non-drilled seeds with and without fungicide treatment had nearly perfect germination percentages, as did drilled seeds with fungicide treatment (98%), whereas germination rates were slightly reduced without fungicide treatment (~90%). The seed coat is maternal rather than
zygotic and was partially removed by sanding prior to tissue sampling. The provision of direct physical access to embryo tissue differs from some previously published seed DNA extraction methods (13). We saw no significant contamination of flour by seed coat fragments and no maternal skewing of SNP signals. The drill bit used to sample seed tissue was cleaned between each seed by drilling into a pencil eraser. This cleaning precludes significant cross contam- ination (Figure 1E). A modified 96-well plate was a useful holder for seeds during the tissue sampling process. Holding seeds by hand, tweezers, and most other devices is cumbersome and inefficient, so this plate- based seed tissue sampling approach sped up the whole DNA extraction process signifi- cantly compared with single-seed handling on a flat, concave, or indented surface (18). As reported previously, microwell plates are convenient for storage of suitably small seeds (13,18). By using the same modified plate to hold seeds during sampling and storage, we avoided the need to transfer seeds and minimized chances for loss of seed identity. Undelinted cotton seeds will fit more snugly in the plate, provided they are not too large. Large seeds and seed size variation can cause some problems, but these issues can be addressed by applying extra pressure to force a larger seed into a well or applying water-soluble glue to the back of the plate for securing seeds too large or too small to fit snugly into the well. DNA yields varied somewhat from seed to seed but were sufficiently consistent to use for large-scale PCR-based genotypic screening. We suspect that most variation in DNA yields arose from differences in the amounts of tissue obtained per seed and the relative particle size distributions, where finer particles would be expected to yield more DNA. It is also possible that differ- ences in seed composition also affected DNA extraction efficacy.
242 DNA from both seeds and cotyledon
extracts was tested and found to be ampli- fiable more than 1 year after extraction and amenable to other PCR reactions (e.g., for SSR markers) (Supplementary Figure 1). Seed viability was not affected for at least 1 year based on our tests. Thus, the user needs neither to germinate the seed quickly nor analyze the DNA quickly. However, fungicide treatments were beneficial to germination rates. In summary, we report very simple proce-
dures for non-destructive DNA extraction and PCR-based genotyping of individual cotton seeds and seedlings. The extraction methods are sufficiently cost-effective and time-efficient to use on a large scale, and are reliable enough for rigorous situations. They are not intended for setting up sequencing libraries. The main cost is for the microwell plates and totals less than $0.05/sample. Drilled seeds can germinate, but protection by a fungicide can increase germination rates. Up to 20,000 seeds can be processed and genotyped in 3 weeks by a single person, and many more seedlings can be processed in a similar amount of time, perhaps up to 10-fold. By extracting DNA and genotyping cotton seed or seedling cotyledon tissue instead of leaf tissue, these methods make it feasible for small laboratories to apply large- scale genotyping and MAS before planting, transplanting, or crossing, and thus facilitate PCR-based assays in the course of daily operations.
Author contributions
X.Z. was involved in the experimentation, analysis of data, and drafting and editing of the manuscript. K.A.H. was involved in the conceptual development, surveying extraction methods, and early testing. A.B.V.M. was involved in the experimentation and analysis of data. F.W. was involved in the experimentation. D.M.S. conceived the project and contributed to the experimental design, manuscript preparation, editing, and submission. R.L.N. and D.C.J. gave advice and helped revise the manuscript.
Acknowledgments
We thank Dwaine A. Raska for assisting in field and greenhouse research. We acknowledge partial support from the following sources: Texas A&M AgriLife Research, Cotton Incorporated, and the Texas State Support Committee. We grate-
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