Genome-wide quantification of homeolog expression ratio revealed nonstochastic gene regulation in synthetic allopolyploid Arabidopsis

Abstract

Genome duplication with hybridization, or allopolyploidization, occurs commonly in plants, and is considered to be a strong force for generating new species. However, genome-wide quantification of homeolog expression ratios was technically hindered because of the high homology between homeologous gene pairs. To quantify the homeolog expression ratio using RNA-seq obtained from polyploids, a new method named HomeoRoq was developed, in which the genomic origin of sequencing reads was estimated using mismatches between the read and each parental genome. To verify this method, we first assembled the two diploid parental genomes of Arabidopsis halleri subsp. gemmifera and Arabidopsis lyrata subsp. petraea (Arabidopsis petraea subsp. umbrosa), then generated a synthetic allotetraploid, mimicking the natural allopolyploid Arabidopsis kamchatica. The quantified ratios corresponded well to those obtained by Pyrosequencing. We found that the ratios of homeologs before and after cold stress treatment were highly correlated (r = 0.870). This highlights the presence of nonstochastic polyploid gene regulation despite previous research identifying stochastic variation in expression. Moreover, our new statistical test incorporating overdispersion identified 226 homeologs (1.11% of 20 369 expressed homeologs) with significant ratio changes, many of which were related to stress responses. HomeoRoq would contribute to the study of the genes responsible for polyploid-specific environmental responses.

Figure 1.

Overview of the quantification of the expression level ratio of homeologs. (A) The generation of synthetic allopolyploid mimicking A. kamchatica. HomeoRoq was applied to this species. (B) Homeolog discriminative RNA-Seq pipeline. Genome/gene sequences of parental species are individually assembled. RNA-seq reads observed from the target allopolyploid are mapped onto both H- and L-genomes independently. Based on the number of mismatches, genomic origin of the reads is classified.

Figure 2.

A visualization example of mapped reads on H-genome. H-origin reads are perfectly matched to the H-genome, while L-origin reads have a few mismatches to the H-genome. This confirms that the proposed method estimate genomic origin successfully.

Figure 3.

Comparison of homeolog expression ratios between the bioinformatics analysis and Pyrosequencing. The homeolog expression ratios of five genes estimated by HomeoRoq and Pyrosequencing (PyroMark) are compared. The data are shown for three individuals at control (ctrl) and stress conditions.

Figure 4.

The ratio of homeolog expression level. (A) Histogram of expression ratio in control condition. (B) Histogram of expression ratio in stress condition. (C) Scatterplot of the ratio of homeolog expression level between two conditions. Each dot corresponds to one homeologous pair. Red dots indicate homeologs whose expression ratio was significantly changed over the conditions. (D) Scatterplot of the ratio of homeolog expression levels. Red dots indicate homeolog whose expression ratio was regarded as significantly changed over conditions with Fisher’s exact test.

Figure 5.

Changes of homeolog-specific expression levels. X-axis and Y-axis are RPKM of H- and L-origin expressions, respectively. Gray dots represent expression levels of the entire homeologs in control environment. Each arrow from a black point means the expression change by the cold stress. The tip of the arrow indicates the expression level of the gene after the cold stress.