Study On Molecular Mechanism Of S1-mediated Interspecific Hybrid Sterility For Asian And African Rice

Hybrids of Asian rice?Oryza sativaL.?and African rice?Oryza glaberrima?have strong hybrid vigor,but often suffer from severe hybrid sterility,which hampers the utilization of heterosis in rice.The S1 locusmapped to a 29-kb on chromosome 6 is one of an important genetic factor causinghybrid male and female sterility in S1heterozygotesofAsian and African rice,but the genefor S1-mediated hybrid sterility and underlying molecular genetic mechanismremain unclear.In this study,predicted candidate open reading frames?ORFs?in S1locus were analyzed by multiple assays such as CRISPR/Cas9-based genome editing system,functional complementary and yeast two-hybrid assay.The OgTPR1 has been cloned as key facor for S1-mediated hybrid sterility.The main results are as follow:1.The common allelicgenes of S1 locus in Asian and African rice,OsTP1,OgTPR1,OsORF7,and Og ORF7 were knocked-out by C RISPR/Cas9-based genome editing systemAnd the resultant mutants show a full fertile phenotype indicating that these genes were not require for the gametogenesis.2.The osorf7,ostp1 and ogorf7,ogtpr1 mutants were crossed with RP-s or NIL-g,respectively.Among the four mF₁ populations,only the mF₁derived from the corssing of ogtpr1and RP-s showed full fertile,indicating that OgTPR1is required for the S1-mediatedinterspecific hybrid sterility.3.OgTPR1 complementation assays showed that transformation ofOgTPR1 alone could not cause sterility in RP-s.The result suggested that S1 hybrid sterility requires other African rice specific factorsbesides OgTPR1 in hybrids.4.Moreover,the semi-sterility phenotype of RP-s/tOgTPR1 crossed withogtpr1 showed that transgenic OgTPR1 could rescue S1-mediated hybrid sterility inN IL-g-ogtpr1 knockout plants.This result further supported that S1-mediatedinterspecific hybrid sterilityrequired other specific factors from African rice.5.To identify the specific factors from African rice,specific genes expressed inthe S1locusof African rice,Og ORFa2^a6,were knocked-out by C RISPR/Cas9-based genome editing system.The results showed that Og ORFa5is not required for the S1-hybrid sterility.Analysis of the other genes for their roles in S1-hybrid sterility is under way.6.Yeast two-hybrid assays indicated that OgO RFa6 can not interact with OgTPR1in full-length,but the second trypsin-like peptidase domain of OgTPR1.This study confirms that OgTPR1 isone of the essential genes forS1-mediatedinterspecific hybrid sterility.And this study provided a solid genetic evidence for identifying other candidate genes from African rice in S1 locus.