Morphological Observation And Gene Mapping Male Sterile Dmtl Mutant In Rice (Oryza Sativa L.)

Rice(Oryza sativa L.)is one of the most important crops for humans.Male sterility is a phenomenon that does not form viable pollen but normal pistil due to abnormal or degenerative in male organ.Male sterility is at the core of the utilization in rice hybrid breeding and heterosis.and it’s significant in safeguarding national food security.Researching on the related mechanisms of rice male sterility has a certain theoretical and practical significance.Through the morphological analysis and gene mapping,the main results are as follows:1.The dmt1(defective microsporocyte tapetum1)is a stably male sterility mutant,obtained by irradiation of 60Coγ rays in songxiangzaogeng.The dmt1 was crossed with the parent songxiangzaogeng,and the F1 phenotype was consistent with the wild type.In the F2,the fertile and sterile lines showed a typical 3:1 separation,indicating that the dmt1 phenotype was controlled by a mononuclear recessive gene.Compared with the wild type,the dmt1 did not show obvious phenotypic defects during vegetative growth.In the reproductive growth period,despite the normal growth of its inflorescence and flower organ,no obvious phenotypic defects in the shape of the spikelets and the number of floral organs,but their anthers were small and white.The I2-KI staining test showed that the dmt1 mutant had less pollen and wrinkled,and were not stained.DAPI staining revealed that dmt1 could undergo normal meiosis to release microspores.Paraffin section and transmission electron microscopy analysis showed that the dmt1 appeared to be degraded earlier from the microspore stage.The main manifestation was that dmt1 mutant had lighter tapetum staining and more vacuoles,the organelles of the carpet cells are dissolved,with dispersed contents,and reduced phlegm,as well as damaged pollen wall;With the continuous development of the anthers,the vacuolar microspores in the wild type begin to accumulate starch and finally form mature pollen grains.However,the tapetum and microspores of the dmt1 mutant continue to degrade,leaving the atrophic microspore cavity.Morphological analysis showed that DMT1 mainly affected the development of tapetum,microspores and pollen wall after meiosis in rice anthers.2.To clone the DMT1 gene,the dmt1 was crossed with the Nipponbare,and a F2 mapping population was constructed.The existing rice genome sequence information was used to screen for polymorphic primers.We found that the target gene was closely related to syrb3-7c on chromosome 3 by BSA method.Linkage and further confirmation by F2 plants.To further fine mapping DMT1,a series of polymorphic markers were developed near the mark syrb3-7c,and polymorphic marks were developed by re-sequencing two parents.Finally,the DMT1 gene was mapped between syrb3-7c1-43 and syrb3-7c1-6 on chromosome 3,with a physical distance of 512 kb.3.In order to investigate the developmental regulation network of DMT1 gene that may participate in rice anthers,we analyzed the expression of some rice pollen wall and tapetum development genes in dmt1 mutants by semi-quantitative RT-PCR.The expression of four pollen wall development genes CYP703A3-3,CYP704B2,WDA1 and DPW2,also,the four tapetum development genes EAT1,DTC1,DTM1 and GAMYB changed significantly,indicating that DMT1 gene may plays an important role in the development of rice anthers.