PMC

Scatter plots showing correlation in microarray gene expression levels based on linear regression of (a) Ae. tauschii mean fluorescence intensities vs. T. turgidum mean fluorescence intensities, (b) T. turgidum mean fluorescence intensities vs. T. aestivum mean fluorescence intensities, and (c) T. aestivum mean fluorescence intensities vs. MPV_1:1 model estimates.

cDNA–SSCP analysis of six genes with nonadditive expression levels based on microarray experiments. Assays were performed on three transcripts that were classified as upregulated (A) and three transcripts that were classified as downregulated (B) in synthetic T. aestivum compared to MPV_1:1 values. (A) TC252860 corresponds to oligonucleotide feature FGAS.02071 that was expressed 2.3-fold higher in synthetic T. aestivum. TC267682 corresponds to oligonucleotide feature FGAS.03312 that was expressed 3.7-fold higher. TC262784 corresponds to oligonucleotide feature FGAS.00736 that was expressed 2.6-fold higher. (B) TC267455 corresponds to oligonucleotide feature USDAWHE.02876 that was expressed 3.4-fold lower in synthetic T. aestivum. TC267082 corresponds to oligonucleotide feature FGAS.02280 that was expressed 3.0-fold lower in synthetic T. aestivum. TC238480 corresponds to oligonucleotide feature USDAWHE.05946 that was expressed 2.7-fold lower. Arrows indicate differential expression of homoeologs. Two independent replicates were in agreement; only one is pictured for the homoeoloci in each group. Images of both single-stranded and heteroduplex conformations are shown for products of cDNA amplification with TC267682 and TC267082.

Microarray experimental design. RNAs from each of three biological replicates of genotypes T. turgidum (AB genome), Ae. tauschii (D genome), and their derived synthetic hexaploid T. aestivum (ABD) were used in microarray hybridation experiments on a spotted 70-mer oligonucleotide array. Rather than mixing RNAs from Ae. tauschii and T. turgidum to create an in vitro synthetic for competitive hybridizations against synthetic T. aestivum, each parental line was independently cohybridized and later averaged to estimate mid-parent expression levels. For treatment 1 (Trt 1), Ae. tauschii transcripts were hybridized against T. turgidum transcripts; for treatment 2 (Trt 2), Ae. tauschii transcripts were hybridized against T. aestivum; for treatment 3, T. turgidum transcripts were hybridized against T. aestivum. Three biological replicates were used per treatment for a total of nine slides. Each feature is replicated on the 17K microarray, as indicated by the nearly identical hybridization patterns observed on a sample of our experimental slides.

(A) cDNA–SSCP analysis of cDNA–AFLP differentially expressed transcript AFLP-23. Although total AFLP-23 transcript levels are similar between diploid Ae. tauschii, tetraploid T. turgidum, and the derived synthetic T. aestivum, homoeologous transcripts are differentially expressed in seedling leaves following polyploidization (). Equal amounts of second-strand cDNAs from Ae. tauschii (D), T. turgidum (AB), and synthetic T. aestivum (ABD) were PCR amplified with AFLP-23 primers and run on MDE polyacrylamide gels. The cDNA–SSCP technique was suitable to identify suppression of a T. turgidum homoeoallele (arrow). (B) Images of the four differentially expressed homoeologous transcripts identified by cDNA–SSCP analysis of 30 arbitrary homoeologs. Equal amounts of second-strand cDNAs from Ae. tauschii (D), T. turgidum (AB), and synthetic T. aestivum (ABD), as well as an in vitro synthetic control (AB + D cDNAs equally mixed) were PCR amplified with conserved primers for each locus and run on MDE polyacrylamide gels. TC270558 was run prior to streamlining the procedure by equally mixing RNAs from three biological replicates (due to lack of observable differences between replicates). Arrows indicate homoeoalleles with differential expression.