Evan Braithwaite and Eric Jellen, Plant and Wildlife Sciences

Introduction

Grains that are high in soluble hemicellulose fiber, such as oat, provide numerous health benefits to consumers. These benefits include decreased risk of heart disease and lowered cholesterol. For this reason, information regarding genes that are involved in regulating starch and fiber synthesis in oat is highly valuable to breeders and cereal companies. However, because of the polyploid nature (allohexaploidy) and intrinsic complexity of the oat genome, given its propensity for chromosomal rearrangement, genetic data available for use in further research is limited, with many important regions still unsequenced. One such region is the gene coding for adenosine diphosphoglucose pyrophosphorylase (ADPG-PPase). ADPG-PPase is one of the major enzymes involved in oat hemicellulose and soluble fiber synthesis pathways. It acts as a catalyst for the first committed step in the synthesis of ADP glucose, which is then vital in the elongation of cellulose.

Objective

Our goal was to characterize the ADP glucose pyrophosphorylase gene in multiple lines of Avena sativa L. (oat) in order to better understand oat starch biosynthetic pathways and generate molecular markers associated with qualitative and quantitative variation in hemicellulose and starch contents in this cereal. These data would have eventual applications in creating higher soluble-fiber oat lines for consumers.

Methodology

Oat from each of the cultivars we aimed to sequence (HiFi, Sol-Fi, and three diploid cultivars) was grown in the greenhouse and DNA was extracted. Primers were designed from expressed sequence tags and used to amplify full-length copies of the gene. Cloning and Sanger sequencing techniques were used to generate sequencing data in each line. Geneious software has been used to align the sequences and to begin the process of determining the size and location of introns, identification of SNPs, and comparison of the sequences. In HiFi, the gene was sequenced in 3 overlapping fragments, but in the other cultivars only 2 overlapping fragments were required.

Results and Discussion

Sequences were successfully generated for the ADP-Glucose Pyrophosphorylase gene. The consensus sequence in the SolFi line was found to be 4.8 kb. The coding sequence for this gene was 1.8 kb, leaving 3kb of intron regions. Further expression analysis and variant comparison will be done to identify molecular markers and understand the relation of this gene to starch phenotypic outcomes in each of the cultivars. I was able to participate in the early and mid stages of this project, and contributed to the generation of a portion of the sequencing data. However, over the summer I was given the opportunity to complete a research internship at the Max Planck Institute in Germany, and was unable to continue working on this project as previously expected. My advisor determined that it would be better to allow other students to finish this project in my absence.

Conclusion

It has been exciting to be involved in this field of research. Although a better understanding of starch synthesis pathways shows promise and will likely lead to increased nutritional benefits and human health outcomes, such results are still elusive due largely to the difficult nature of sequencing and analyzing complex polyploid genomes such as oat. I am grateful for the opportunity to participate in such a challenging project and anticipate continued use of the skills and techniques I have learned while doing so.

References:

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