Comparative cytological and transcriptome analysis reveals high pollen fertility and upregulation of environmentally sensitive genic male sterility genes in neo-tetraploid rice

Abstract

Background: Autotetraploid rice is a useful germplasm for polyploid rice breeding; however, low seed setting is a major hindrance for the utilization of autotetraploid rice. Our previous study demonstrated that neo-tetraploid rice have great yield potential, which is thought to be one effective way to overcome the low fertility of autotetraploid rice. However, there is little known about the cause of high pollen fertility in neo-tetraploid rice. Here, we employed cytology and RNA-seq to study the molecular genetic mechanism of high pollen fertility in neo-tetraploid rice. Results: Cytological observations indicate that H1 displayed high pollen fertility (95.62%), lower percentage of pollen mother cells（PMCs）abnormalities, and stable chromosome configurations during the pollen development process compared with its two parents. RNA-seq analysis detected 440 differentially expressed genes (DEGs) in neo-tetraploid rice compared with its two parents. Of these DEGs, 193 were annotated as pollen fertility-related genes, and 129 (~66.8%) exhibited significant upregulation in neo-tetraploid rice compared with its two parents, including nine cloned genes ( TMS9-1 , TMS5 etc.) that were validated by qRT-PCR and had been demonstrated to be pollen fertility-related genes. We further selected TMS9-1 and TMS5 as the candidate gene and analysed its pollen fertility in neo-tetraploid rice using the CRISPR/Cas9 technique. Significant variations have been detected in pollen fertility value, pollen development process and expression level in H1 and its knock out lines. Conclusion: Our finding provides strong evidence for the regulatory mechanisms of neo-tetraploid rice, and upregulation of pollen fertility-related genes should be associated with high fertility. Moreover, knockout of environmentally sensitive genic male sterility genes in the present study provides the new useful germplasm for polyploidy rice breeding.