REPORTS A B
Figure 4. Seed germination test after non-destructive tissue sampling for DNA extraction. (A) Image of tissue-sampled seeds after germination in a “ragdoll.” (B) Image of seedlings from sampled seeds grown in soil cups; cotyledons lack areas of marginal tissue due to previous sampling from the seed before germination.
plate reader (BMG LABTECH GMBH, Cary, NC). The plate reader data were imported into KlusterCaller software (LGC Genomics) for genotyping. In the event that greater ampli- fication was required, plates were returned to the Hydrocycler (LGC Genomics) for itera- tions of three additional PCR cycles, with the denaturation steps at 94°C for 20 s followed by annealing at 57°C for 1 min.
SNP amplification from cotton seed DNA The protocols above for non-destructive extraction of DNA from cotton seeds and SNP genotyping were tested with experi- mental research materials. DNA samples extracted in the prescribed manner from 88 first-generation backcross (BC1F1) cotton seeds were placed into the first 11 columns of a 96-well plate, where these 88 seeds were known on the basis of pedigree to be segregating for SNP Gl_072641. This is an alien chromosome segment derived from the species Gossypium longicalyx, a wild African relative of cultivated cotton. Column 12 of the same 96-well plate was dedicated to conventionally extracted DNA from the parental leaf tissue, using Qiagen DNeasy Plant Mini kits (#69106; Qiagen, Valencia, CA), and two non-template controls. All samples in the plate were amplified using the primer set for SNP Gl_072641.
SNP amplification for cotton cotyledon DNA The protocols above for non-destructive extraction of DNA from cotton seedling
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cotyledons and SNP genotyping were tested with experimental research materials. Using the procedures described above, DNA samples were extracted from 36 s-generation backcross (BC2F1) hybrid seedlings from a G. hirsutum*2/Gossypium barbadense plant known to be heterozygous for an alien segment and then amplified using the SNP Gb_010283 that is located in that segment. DNA was extracted from young leaves of the parents using Qiagen DNeasy Plant Mini kits. The test plate included 2 non-template control wells and 16 empty wells.
Germination of drilled seeds after PCR analysis Drilled seeds were stored in modified plates in cabinet drawers at room temperature in a conventional air-conditioned office (Figure 1C). Germination rates were assessed by placing seeds in ragdolls of germination paper that had been pre-soaked with tap water alone or tap water plus Banrot (#MK03; BWI) (0.5 mL/100 mL tap water). Ragdolls were incubated in the dark at 30°C for 48 h before assessment.
Results and discussion
KASP ratios from the BC1F1 cotton seed DNA samples fell into two well-defined clusters (Figure 2). The patterns and positions of the two clusters of seed-based SNPs were congruent with expectations for a segregating marker with two genotypes: one heterozygous, similar to the F1 hybrid
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parent reference (symbolized as FADD) and one homozygous, similar to the recurrent parent reference (symbolized as AADD). Each seed-based data point fell along the same ray (angle) as the respective reference samples that had the same genotype. The observed ratio in this batch of 88 BC1F1 seeds was 44:44, which happened to adhere exactly to the conventionally expected test cross ratio (1:1). Relative to conventionally prepared DNA
from segregating plants, we noted that the clusters were significantly more dispersed along the ray (angle), which indicates overall fluorescence amplitude was somewhat variable. We typically observed similar levels of dispersion at right angles to the ray, indicating that SNP-specific fluorochrome ratios were similar for the non-destructive seed-based extractions and conventional leaf-based extractions. There would seem to be two likely explanations for the additional variation in overall SNP signal amplitude, one being increased variation in DNA concen- tration for seed-based extractions and the other being variation in compounds that interfere with the PCR process, such as phenolics. Three or six extra PCR cycles were typically needed for DNA samples extracted from seeds compared with those from conventional leaf-based extractions. DNA samples extracted from cotyledons
were run in duplicate wells, and KASP ratios from all of the seedling DNA samples fell into two well-defined clusters (Figure 3). The patterns and positions of the clusters of seed- based SNPs were congruent with expecta-
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