Summary: | Hybrid sterility is the major obstacle to the utilization of inter-subspecific heterosis in hybrid rice breeding. The <i>S5</i> locus, composed of three adjacent genes <i>ORF3</i>, <i>ORF4</i>, and <i>ORF5</i>, plays a crucial role in regulating <i>indica</i>/<i>japonica</i> hybrids’ female sterility. Through a series of crosses involving 38 parents, three alleles of <i>S5</i>, <i>ORF3</i>+<i>ORF4</i>−<i>ORF5n</i>, <i>ORF3</i>+<i>ORF4</i>+<i>ORF5n</i>, and <i>ORF3</i>−/<i>ORF4</i>−/<i>ORF5n</i>, all could be regarded as wide-compatibility alleles, and when crossed with <i>indica</i> or <i>japonica</i> rice, they all showed significantly high fertility. Then, in order to explore the genes’ function, we further knocked out genes by using CRISPR/Cas9-based genome editing. Our results demonstrate that the <i>ORF3</i>+ was not just the protector in the killer-protector system, and knocking out <i>ORF3</i> of the <i>indica</i> allele seriously affected the rice’s normal development. We observed the concrete enhancing hybrid spikelet fertility from the crosses between the <i>ORF4+</i> knockout <i>japonica</i> materials with <i>indica</i> varieties. By conducting the comparative RNA-Seq analysis of young spikelets, we found that the <i>ORF4</i>+/<i>ORF4</i>− could modulate the hybrid fertility by affecting the expressions of genes related to the function of the Golgi apparatus. This study indicated that knocking out the <i>ORF4</i>+ of the <i>japonica</i> allele or using the alleles carrying <i>ORF5n</i> would provide effective approaches to overcome <i>indica</i>/<i>japonica</i> hybrid female sterility in rice breeding.
|