Wild type in Mo17 X H95 background in 22 degree, replicate 2
Wild-type (WT) and isogenic Rp1-D21 mutant plants in B73 × H95 and Mo17 × H95 backgrounds were used for RNA extraction for RNA-seq analysis. The only substantial genetic difference between the isogenic pairs was at the Rp1 locus where the wild type isoline was heterozygous for the H95 and B73 or Mo17 haplotypes while mutant lines carried the Rp1-D21 haplotype instead of the H95 Rp1 haplotype. All the maize lines were grown in constant 22 °C with 12h-day/12h-dark. The 3rd true leaves of 5 individual seedlings collected from 18-day old WT or Rp1-D21 mutant plants from B73 × H95 or Mo17 × H95 backgrounds were pooled for total RNA isolation. RNA concentration and quality were monitored by the NanoDrop and agarose gel electrophoresis. mRNA was isolated from the total RNA by Dynabeads oligo(dT) (Life Technologies) following the manufacturer’s directions. RNA-seq libraries were constructed according to the TruSeq RNA Sample Prep v2 LS protocol (Illumina). Libraries were sequenced using the Illumina HiSeq 2000. Two biological replicates, each consisting of 5 individual plants, and two technical replicates (two lanes) were performed, with 100 nt single end reads. The sequences were aligned to maize genome sequence v2 (www.maizegdb.org) via TopHat  2.0 (TRAPNELL et al. 2009), using all the default parameter settings. The raw counts per gene were calculated using in-house scripts. Only reads with unique alignments were maintained for subsequent analyses. Genes with total read counts less than 20 were filtered out. Differentially expressed genes (DEGs) were identified using the software package edgeR  from the Bioconductor suite (ROBINSON et al. 2010). To account for multiple testing, the procedure of Benjamini and Hochberg (1995)for controlling the false discovery rate (FDR) was applied using a threshold of q<=0.05 to determine significance.  BW: Wild type in B73 × H95; BM: Rp1-D21 mutant in B73 × H95; MW: Wild type in Mo17 × H95; MM: Rp1-D21 mutant in Mo17 × H95.