Fig 1.
The first PCR is a multiplex and amplifies the different target regions containing SNP(s). The locus-specific forward primers were tailed with an M13 sequence at the 5’-end and the reverse primers were tailed with Ion truncated P1/B sequence. The generated amplicons contains an M13 tail and Ion truncated P1/B sequence. The second PCR involves the addition of barcodes and standard Ion A adapter to the amplicons.
Fig 2.
Average number of reads per marker analyzed using non-normalized (x-axis) and normalized (y-axis) DNA.
The top figure used 100 nM primers for PCR and the bottom figure used 12.5 nM primers for PCR.
Fig 3.
Average number of sequence reads per marker using 17 ng (x-axis) and 141 ng DNA (y-axis) as template.
The top figure used touch down (TD) and the bottom figure used non-TD PCR. The arrow points to the number of reads of CNL9 marker for Sr35.
Fig 4.
Average number of reads per marker as affected by low (y-axis) or high primer concentrations (x-axis).
The top figure used normalized and the bottom figure used non-normalized DNA.
Fig 5.
Average number of reads as affected by different primer concentrations: 6.25 nM, 12.5 nM, 2 tiers of 2 nM and 25 nM, and 3 tiers of 2 nM, 15 nM and 30 nM.
Standard errors are shown as error bars on top of columns.
Fig 6.
Average number of reads per marker in non-TD (blue column) and TD PCR (red column).
Standard errors are shown as error bars on top of columns. Samples were run using the primer combination of 8 nM and 16 nM.
Fig 7.
Minimum percentage of favorable allele reads from three GBMAS libraries constructed using the 2-tier primer pool of 8 nM and 16 nM primers and non-TD PCR.
Table 1.
Primer specificity and theoretical read percentages of expected favorable SNP allele (A) amplified from one to three genomes of wheat.