An efficient experiment design helps to identify differential expressed genes (DEGs) in RNA-seq data in studies of plant qualitative traits

1 In this study, we conducted comparative transcriptome analysis between homozygous dominant 2 parent and heterozygous F1 hybrid with homozygous recessive parent in qualitative trait study of 3 common wheat (Triticum aestivum L.). Two sets of near-isogenic lines (NILs) were used: one set of 4 NILs carrying powdery mildew resistance and susceptible Pm2 alleles, the other set of NILs carrying 5 different awn inhibition gene B1 alleles. The results demonstrated that 2,932 DEGs were identified 6 between L031 (Pm2Pm2) and Chancellor (pm2pm2), while 1,494 DEGs presented between F1 hybrid 7 (Pm2pm2) and Chancellor, the co-regulated DEGs were 1,028. For the wheat awn inhibition gene B1 8 test, 720 DEGs were identified between SN051-2 (B1B1) and SN051-1 (b1b1), and 231 DEGs were 9 identified between F1 hybrid (B1b1) and SN051-1, the co-regulated DEGs were 180. Hierarchical 10 clustering analysis of co-regulated DEGs showed that dominant parent and F1 hybrid were clustered 11 as the nearest neighbors, while recessive parent showed an apparent departure. The results showed 12 that the overlapping DEGs between dominant parent and F1 hybrid with recessive parent reduced the 13 number of interested DEGs to only one-quarter (or one-third) of that between dominant and recessive 14 parent, these overlapping loci could provide insights into molecular mechanisms that are affected by 15 causal mutations. 16