Assessment of phosphorous deficiency-dependent single-nucleotide polymorphisms in the transcriptome of white lupin

White lupin undergoes a coordinated developmental and physiological response to phosphorus (P) deficiency, which includes the formation of dense, proteoid roots (also known as cluster roots) and the secretion of acid phosphatases and other chemicals that improve the release of free phosphate in soil. A recent transcriptome comparison of white lupin plants grown in either P-sufficient or P-deficient conditions performed in the laboratory of Dr. Carroll Vance at the University of Minnesota identified, surprisingly, nine genes that contained specific single-nucleotide polymorphisms (SNPs) that were present in only one of the two conditions, raising the question of how these differences arise and whether they are regulated by P. I investigated this question by determining whether the single-nucleotide polymorphisms exist in the genome or whether they are introduced during or after transcription. In addition, I used bisulfite sequencing of +P and –P white lupin genomic root DNA to determine if cytosine methylation occurs in a nutrient-dependent manner in these genes, and whether this may be involved in the expression of nutrient-specific SNPs. My sequencing analysis determined that none of the SNPs identified as being differentially present in the +P and –P plants was present in the genomes of these plants. This suggests that the SNPs arose during or after transcription, potentially through a process of RNA editing. Using bisulfite sequencing of one of these genes, I determined that there was no difference in the pattern of methylation between the +P and –P conditions. However, this analysis also revealed an unusual pattern of methylation present in both conditions, as all cytosines on the reverse strand were methylated, while only a small subset was methylated on the forward strand. Finally, my results showed that the sequences flanking the differential SNPs at the -2, -1, +1, and +2 positions were strongly enriched for G and C.