Map-based Cloning And Functional Analysis Of OsMTOPVIB(Oryza Sativa MEIOTIC TOPOISOMERASE VIB-LIKE) In Rice

Meiosis is the key step in sexual reproduction.Meiosis not only keeps the stable transmission of genetic information,but also enhances the genetic diversity of the population through the crossover of non-sister chromatids of homologous chromosomes,which is the basis for biological adaptation and evolution.Meiotic crossover formation is initiated by DNA double stranded break?DSB?formation.However the mechanism underlying DSB formation in rice remains largely unknow.In this study,we isolated a rice mutant,Oryza sativa meiotic topoisomerase VIB-like?osmtopVIB?,which is generated by ⁶⁰Co?-ray radiation.osmtopVIB mutant exhibited both male and female sterile.Genetic analysis showed that the ratio of fertile and sterile plants in the F2 hybrid plants was 3:1,indicating that the mutant phenotype was caused by a recessive single gene mutation.Phenotypic observation showed that the vegetative stage of the mutant was normal.However,after entering into the reproductive stage,although the floral organs were normal,they could not produce fertile pollen grains.Semi-thin sections and DAPI staining showed that the meiotic process in osmtopVIB was abnormal.In the early prophase I,homologous chromosomes could not complete the pairing process.By immunofluorescence staining,it was found that the axial elements REC8,PAIR2 and PAIR3 could be located on the mutant chromosomes but the central element ZEP1 could not be located on the chromosomes,indicating that the mutant could not finish assembly of the synaptic complex.Meiotic recombination is initiated by the induction of DNA double strand breaks?DSBs?.The immunostaining of DSB-specific antibody?H2AX showed that no?H2AX signal was observed in osmtopVIB,indicating a defect in the DSB formation of the mutant.Meanwhile,the homologous recombination marker protein DMC1 and MER3 could not be located in the mutant too,further demonstrating that the homologous recombination was also abnormal in the mutant.Therefore,we think that the loss of OsMTOPVIB led to the defection in the rice meiotic DSB process.The homologous pairing,synapsis and recombination process was also affected which resulted in the mutant sterility.By map-based cloning and next generation sequencing,we found a deletion of one base in the second exon of LOC_Os06g49450 on rice chromosome 6,leading to premature termination of protein translation.Sequence and structural analysis of OsMTOPVIB showed that OsMTOPVIB shares homology with the topo VIB subunit and possesses four typical domains of TOPVIB,including GHKL domain,SmD,transducer domain and C-terminal domain.Further yeast two-hybrid experiments showed that OsMTOPVIB interacted with SPO11-1 and SPO11-4 in rice,respectively,indicating that OsMTOPVIB could combine with SPO11-1 and SPO11-4 to form a complex to catalyze meiotic DSB formation.These results indicate that OsMTOPVIB plays an important role in rice meiosis,and this is the first homologue of topo VIB found in monocotyledonous rice.It provides a new insight for understanding the conserved function of topo VIB in meiotic DSB process.