(A) Genotyping of parental lines and 576 Ae. tauschii F
plants in the AL8/78 × AS75 mapping population with Illumina GoldenGate oligonucleotide assay querying an A/G SNP at EST locus BE499478 (nucleotide position 403 in the Ae. tauschii reference sequence, http://probes.pw.usda.gov:8080/snpworld/Search). The graph is a normalized polar coordinate (R, θ) plot. Theta (x-axis) is an angle of deviation of Cy3 and Cy5 fluorescence from pure Cy3 and pure Cy5 signal (0 and 1). The closer a point is to 0 or 1, the greater the proportion of Cy3 or Cy5 fluorescent signal, respectively, is present. R (y-axis) is the Manhattan distance of observed Cy3 and Cy5 fluorescence to the origin (pole), which is 0 fluorescence. The closer a point is to 0 on the y-axis, the weaker the total fluorescence. The red, blue and purple ovals have the diameter of 2 standard deviations computed from the dispersal of the red, blue and purple dots, respectively. The darker colored regions define genotype call areas for homozygous (red and blue dots) and heterozygous (purple dots) plants. The numbers of plants in each cluster are indicated below the x-axis. (B) Clustering of 57 BAC and BiBAC super-pools assayed with the same oligonucleotides as in (A). Genotype call areas derived from the mapping population shown in 1A were directly exported for analysis of the BAC pools. The green and orange spots represent the parental genotypes of the mapping population; the red spots are the BAC and BiBAC pools scored by us as positive and black spots BAC and BiBAC pools scored as negative. Note that all red spots have the same normalized angular deviation (normalized theta) as DNA of Ae. tauschii AL8/78 and large distance from origin (R value) indicating high fluorescence. In contrast, the black spots have a low R value indicating only residual Cy3 and Cy5 fluorescence and variable values of normalized theta ranging from 0 all the way to 1. (C) Clustering of the same 57 BAC and BiBAC super-pools without defining call areas on the basis of clustering segregating Ae. tauschii SNP genotypes. Note failure of separation of the positive and negative clusters from each other.