Deep Sequencing Of Maize Seedling Transcriptomes To Understand The Impact Of The ROUGH ENDOSPERM3 Splicing Factor

Maize kernel development is of high economic interest, as more acres of corn were harvested in 2011 than any other crop in the United States (USDA NASS, 2012). An understanding of the molecular mechanisms that govern maize endosperm formation will lay the groundwork for improvements in yield and grain quality. The maize rough endosperm3 (rgh3) mutant exhibits defective kernel development. Prior characterization of the rgh3 mutant indicates that the gene is required to repress cell proliferation and promote cell differentiation. Less than 50% of rgh3 kernels germinate, and rgh3 seedlings have adherent leaves and stunted growth, typically dying 15-18 days after planting. rgh3 is a hypomorphic allele of the U2AF³⁵-related protein (ZmURP). In humans, URP is associated with the minor and major spliceosomes, but its biological role is not clear in maize. We hypothesize that rgh3 has an effect on mRNA splicing. To test this, we compared rgh3 and normal sibling transcriptomes in an RNA-seq experiment. Twelve cDNA libraries were constructed from the roots and shoots of three seedlings of each genotype. The libraries were multiplexed and sequenced on one lane of the HiSeq 2000 platform. The run produced 149 million paired-end 100 bp reads that mapped to more than 35,000 genes. mRNA splicing in rgh3 and normal tissues was analyzed using a Mosaik2-GSNAP-Cufflinks pipeline to quantify isoform abundance. Forty-five genes were bioinformatically predicted to have altered splicing in rgh3. Reverse transcription PCR was used to validate intron retention events that were more likely to occur in the rgh3 mutants.