NEW YORK (GenomeWeb) — Researchers from the University of Liverpool have constructed a genome-wide map of methylation across the three wheat subgenomes.
Liverpool's Laura Gardiner and her colleagues used a combination of sodium bisulfite treatment and targeted gene enrichment to tease out methylation sites within the allohexaploid wheat genome. As they reported today in Genome Biology, the researchers found that methylation is highly conserved across all three wheat subgenomes, though there is variation from subgenome to subgenome.
"This work opens up a whole new level of genetic variation which can be exploited by wheat breeders," Gardiner said in a statement. "In the future we see epigenetic marks becoming an important new tool in this area."
The wheat genome can be technically difficult to work with, owing to its immense size and multiple copies, Gardiner added.
To tackle its epigenome, she and her colleagues used a genome-wide methyl-Seq approach in which genomic enrichment was followed by bisulfite treatment and Illumina HiSeq paired-end sequencing. They applied this method to six seven-day old Chinese Spring seedlings that had been grown at either 12° C or 27° C, sequencing them to an average 102X depth across 96.3 percent of the bait sequence.
With MethylKit, they noted a high level of conservation of methylation between the replicate samples.
Because of that, the researchers then pooled those replicates for further analyses, increasing their coverage to an average 297.6X depth and mapping to 97.5 percent of the capture probes.
The researchers disentangled the three wheat subgenomes by relying on homologous SNPs. They noted, however, that about 45 percent of methylated regions were methylated across all three subgenomes.
While each subgenome had about the same level of methylation, there were regions that were only methylated in some of the three subgenomes. Again using MethylKit, Gardiner and her colleagues identified methylation sites that were unique to a certain subgenome, shared by two subgenomes, or shared by all three.
Methylation sites unique to the A subgenome were enriched for genes involved in biosynthesis or metabolic process, while those particular to the B subgenome were enriched for genes with biosynthesis or metabolic processes as well as growth and membrane and cell wall functions. D subgenome methylation sites, meanwhile, were enriched for genes associated with zinc ion transmembrane transport and cellular homeostasis.
They also uncovered regions across the wheat genome that were differentially methylated. In the 12° C samples, they found 11 differentially methylated regions specific to genome A, 15 specific to genome B, and four specific to genome D, while the 27° C samples included seven differentially methylated regions in genome A, 18 in genome B, and five in genome D. These regions are enriched for similar GO terms as the unigenome methylation sites, they noted.
These differences in methylation affected gene expression in wheat, the researchers said. They used the same leaf samples they'd collected for the methylation analysis for RNA-sequencing. Based on probability of positive log ratio values, they found 1,158 genes in genome A, 1,321 in genome B, and 1,290 in genome D that were highly differentially expressed between the 12° C and 27° C samples.
Of the sites that were differentially methylated between the 12°C and 27° C samples, some 63 percent were also differentially expressed, suggesting a link between the differential methylation and expression.
Methylation throughout the wheat genome has been largely stable over evolutionary time, Gardiner and her colleagues added. They compared the methylation state of D wheat subgenome to that of Aegilops tauschii, its likely diploid progenitor that hybridized with the AABB progenitors some 10,000 years ago.
Methylation in the Ae. tauschii genome was similar to what the researchers found across all three wheat subgenomes. Of the 3,336 sites that were methylated in all three wheat subgenomes, methylation at those sites in the Ae. tauschii genome differed less than 5 percent of the time.
There were, however, differences between methylation in the Ae. tauschii genome and the wheat subgenomes when considered individually. Methylation sites in Ae. tauschii differed from subgenome A-specific and subgenome B-specific sites some three-quarters of the time. They only differed from subgenome D-specific methylation sites 13.6 percent of the time, suggesting conservation of methylation over time.
"With the ability to characterize genome-wide patterns of methylation we can now address fundamental questions in wheat, such as the role of epigenetics in the domestication of crops and the stability and long-term function of methylation," said Liverpool researcher and senior author Anthony Hall in a statement. "We can also seek to understand how methylation changes important traits for farmers like disease resistance and yield variability."