Reports Non-destructive high-throughput DNA extraction and


genotyping methods for cotton seeds and seedlings Xiuting Zheng1


, Kevin A. Hoegenauer1 Nichols2 , and Don C. Jones2


1Department of Soil and Crop Sciences, Texas A&M University, College Station, TX and 2 Incorporated, Cary, NC


BioTechniques 58:234-243 (May 2015) doi 10.2144/000114286 Keywords: cotton; seed; seedling; cotyledon; DNA extraction; genotyping Supplementary material for this article is available at www.BioTechniques.com/article/114286.


Extensive use of targeted PCR-based genotyping is precluded for many plant research laboratories by the cost and time required for DNA extraction. Using cotton (Gossypium hirsutum) as a model for plants with medium-sized seeds, we report here manual procedures for inexpensive non-destructive high-throughput extraction of DNA suitable for PCR-based genotyping of large numbers of individual seeds and seedlings. By sampling only small amounts of cotyledon tissue of ungerminated seed or young seedlings, damage is minimized, and viability is not discernibly affected. The yield of DNA from each seed or seedling is typi- cally sufficient for 1000 or 500 PCR reactions, respectively. For seeds, the tissue sampling procedure relies on a modified 96-well plate that is used subsequently for seed storage. For seeds and seedlings, the DNA is extracted in a strongly basic DNA buffer that is later neutralized and diluted. Extracts can be used directly for high-throughput PCR-based genotyping. Any laboratory can thus extract DNA from thousands of individual seeds/seedlings per person-day at a very modest cost for consumables (~$0.05 per sample). Being non-destructive, our approach enables a wide variety of time- and resource-saving applications, such as marker-assisted selection (MAS), before planting, transplanting, and flowering.


In many situations, relatively small numbers of PCR-based markers need to be analyzed across relatively large numbers of individuals, lines, families, or popula- tions. Examples of such PCR-based appli- cations include marker-assisted selection (MAS) for traits that are expensive to determine directly, linkage analysis of localized regions, selection for rare recom- bination events, genetic fingerprint identi- fication of seed or plant specimens, large- scale detection or selection for/against transgenes, and large-scale selection of genotype-specific bi-parental or complex sub-populations to verify or map a quanti- tative locus. Unfortunately, many labora- tories lack the time and resources required


METHOD SUMMARY


Non-germinated seed were organized and immobilized with modified 96-well plates, and small amounts of tissue were obtained by drilling. Tissue from seedling cotyledons was obtained using a hole-punch. Seed or seedling samples were processed in a plate-based format using a low-cost hydroxide-based DNA extraction process. Aliquots were diluted before PCR-based genotyping for SNPs (KASP assays) and simple sequence repeats (SSRs).


Vol. 58 | No. 5 | 2015 234 www.BioTechniques.com


for large numbers of DNA extractions. Methods for rapid extraction of plant DNA have been reported, but almost all involve tissue from whole plants (1–4). In addition, several in-house DNA extraction protocols have been described for temperate crops, based on the use of 96-well microwell plates (5,6). Like a number of other species, DNA


isolation from cotton (Gossypium spp.) is complicated by the presence of phenolic compounds (7). During tissue homoge- nization, such phenolic compounds can oxidize and irreversibly bind with proteins and nucleic acids (8). This irreversible binding produces a gelatinous material, which is hard to separate from organelles


and renders the DNA unsuitable for PCR or restriction enzyme digestion (9). Previ- ously developed protocols for extraction of cotton DNA suitable for PCR use leaf tissue as the starting material and also involve the use of several reagents (7,10,11). A plate-based high-throughput DNA extraction method that relies on young cotton leaf tissue was reported recently by Xin et al. (12), which improved the rate of plant-based DNA extraction but did not address the practical need of many researchers to genotype seeds and/or seedlings. Moreover, DNA extraction from seeds would be especially attractive if it were non-destructive, allowing genotyping and marker-assisted selection (MAS) to be


Cotton , Andrea B. V. Maeda1 , Fei Wang1 , David M. Stelly1 , Robert L.


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60  |  Page 61  |  Page 62  |  Page 63  |  Page 64  |  Page 65  |  Page 66  |  Page 67  |  Page 68