A low-coverage 3′ RNA-seq to detect homeolog expression in polyploid wheat

Although allopolyploid species are common among natural and crop species, it is not easy to distinguish duplicated genes, known as homeologs, during their genomic analysis. Yet, cost-efficient RNA sequencing (RNA-seq) is to be developed for large-scale transcriptomic studies such as time-series analysis and genome-wide association studies in allopolyploids. In this study, we employed a 3′ RNA-seq utilizing 3′ untranslated regions (UTRs) containing frequent mutations among homeologous genes, compared to coding sequence. Among the 3′ RNA-seq protocols, we examined a low-cost method Lasy-Seq using an allohexaploid bread wheat, Triticum aestivum. HISAT2 showed the best performance for 3′ RNA-seq with the least mapping errors and quick computational time. The number of detected homeologs was further improved by extending 1 kb of the 3′ UTR annotation. Differentially expressed genes in response to mild cold treatment detected by the 3′ RNA-seq were verified with high-coverage conventional RNA-seq, although the latter detected more differentially expressed genes. Finally, downsampling showed that even a 2 million sequencing depth can still detect more than half of expressed homeologs identifiable by the conventional 32 million reads. These data demonstrate that this low-cost 3′ RNA-seq facilitates large-scale transcriptomic studies of allohexaploid wheat and indicate the potential application to other allopolyploid species.