Comparative Analysis of Homoeoallele Expression in the Tocol Biosynthetic Pathway during Oat Seed Development

Oats are a rich source of compounds that collectively constitute vitamin E, the tocols. Significant attention has been given to the health benefits of tocols in oats, but little is known about the molecular control of their accumulation during grain development. Next generation sequencing provides an opportunity to explore transcriptomes and thus examine this issue, but challenges remain for differentiation of homoeoalleles and paralogs, particularly in polyploid organisms such as oat with no supporting genome sequence. We sequenced cDNA from 4 stages of oat seed development using Illumina HiSeq 2000 and Roche GS FLX+ technologies. After strict quality filtering including removal of primers, duplicates and short reads, 32 million 100-bp paired-end Illumina and 869,281 Roche reads remained. These high-quality reads were mapped to known genes in the tocol biosynthetic pathway of Brachypodium, barley and wheat. This permitted us to infer that 3 homoeoalleles for each of the 7 tocol biosynthetic pathway genes studied, GGR, HGGT, HPPD, VTE1, VTE2, VTE3, and VTE4, were expressed. Using information from the mapped reads, primers were designed to define coding sequences of the 3 homoeoalleles of these genes by Sanger sequencing. The complete coding sequences of the oat tocol genes are being used to examine the relative contribution of different homoeoalleles to tocol biosynthesis during the course of oat seed development. These findings will advance our understanding of the regulation of tocol synthesis and accumulation in developing oat seeds, and provide a technical strategy for unraveling differential expression of duplicated genes in other organisms.