REPORTS
Solutions and buffers Extractions relied on the basic and acidic buffers described by Xin et al. and von Post et al. (12,18). Buffer A (100 mM NaOH, 2% Tween 20) must be made fresh from 10 M NaOH and 20% Tween 20 stock solutions (#S5581, #P-7949; Sigma-Aldrich, St. Louis, MO) just before use. Buffer B [100 mM Tris-HCl (#BFH0312; VWR, Sugar Land, TX), 2 mM EDTA] should be adjusted to pH 2.0, but does not have to be made fresh for each extraction and can be stored at room temperature.
Primers Two cotton SNPs were used in this study, one for testing seed-based DNA extraction and the other for cotyledon-based extraction. The SNPs were chosen from subsets of large numbers of interspecific SNPs identified by comparative RNA-Seq analysis and experimentally tested using KASP assays (LGC Genomics, Beverly, MA) as part of the computational pipeline evalu- ation process for identifying putative SNPs between cotton (G. hirsutum) and several related 52- and 26-chromosome species (19). SNP Gl_072641 can be detected by KASP assays using the common reverse primer TGTGGAGGCATAGTGAGAGG and forward SNP-specific primers GGTGTAT- GTAAAAGTCCGAAAGCA and GTGTATG- TAAAAGTCCGAAAGCG. SNP Gb_010283 can be detected by KASP assays using the common reverse primer AGATTGACTC- GGGACTTCCT and forward SNP-specific primers CCCCTCATGTTTCTAACTATTTGT and CCCTCATGTTTCTAACTATTTGC.
PCR conditions Two microliter aliquots of DNA extract were added to each well, and the plate was briefly centrifuged before drying down in a bench-top oven for 1 h at 65°C. An 8 µl PCR mixture containing 4.0 µl Reaction Mix (KASP Master mix, #KBS-1016–002; LGC Genomics), 3.826 µl sterile deionized water, 0.11 µl of Assay (Primer) Mix, and 0.064 µl 50 mM MgCl2
were added to each well. The PCR
Figure 3. SNP genotyping of DNA extracted from individual cotton seedling cotyledons af- ter 44 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 conven-
tionally extracted DNA samples are denoted by the symbols * and +. Wells containing non-template controls (no sample DNA) are black, and empty wells are orange. The upper 72 wells contain 36 duplicated samples from the seedling-based DNA extraction meth- od. The 36 seedlings were part of a backcross family segregating for the SNP Gb_010283.
Vol. 58 | No. 5 | 2015 238
for the KASP assays included an acclimation step of 94°C for 15 min. Each of the first 10 cycles included denaturation at 94°C for 20 s, followed by annealing, starting at 65°C for 1 min and then decreasing 0.8°C per cycle to an annealing temperature of 57°C for the final cycle. This was followed by 28 cycles of denaturation at 94°C for 20 s, and annealing at 57°C for 1 min. Plates were then briefly centrifuged and read on a PHERAstar Plus
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