Cytological Observation Of Rice Semi-sterile Mutants And Cloning Of Related Genes

The normal development of female gametes in rice is the premise of normal fruiting.The abnormal development of female gametes in rice will lead to a significant reduction of rice yield.Therefore,the study on the sterility of female gametes in rice is of great practical significance.In this study,we used EMS to induce"Yuefeng B"to obtain the semi sterile mutant m21.The material has normal vegetative growth,normal appearance and structure of flower organs,but the natural seed setting rate is 53.52%±2.In this study,we carried out saturation assisted pollination experiment on the semi sterile mutant m21,and constructed BC₁F₄ and BC₁F₅ backcross lines of the semi sterile mutant m21 for pollen fertility test,pollen vitality identification and mutation Observation on the development period of somatic embryo sac.At the same time,the BSA method based on high-throughput sequencing was used to analyze the related genes.The main results are as follows:1:Using wild-type'Yuefeng B'pollen as supplementary pollination for 3 consecutive days,the seed setting rate of the semi sterile mutant m21 was not significantly higher than that of the self pollination of the semi sterile mutant m21.The results showed that the Semisterility of the mutant was affected by pistil group.2:The BC₁F₅ backcross line of the semi sterile mutant m21 was constructed.A semi sterile line wzn58 with an average fertility of only52.30%±2 was isolated from the BC₁F₅ backcross line,which was similar to the wild type"Yuefeng B",and its pollen fertility was identified by wzn58,The fertile pollen of WZN58 was 87.16%±1 and the germination rate of pollen in vitro was 72.1%±2.There was no significant difference between WZN58 and the wild type"Yuefeng B"pollen in fertility and vigor.It indicated that the pollen fertility and activity were normal.3:Cytological observation on the development process of female gametes in WZN58 showed that about 50%of embryo sacs began to appear abnormal at the early stage of sporogonium development.When sporogonium cells were differentiated,a number of sporogonium like cells were produced,followed by a number of cells with similar morphology to megaspore cells.In the middle stage of development,50%of embryo sacs differentiated into three megaspores,followed by two types of abnormalities,one is three megaspores The spores degenerated gradually from the chalazal end,and finally all degenerated,leaving only the vestiges of degeneration;the other one was the chalazal end degenerated,and finally formed the mononuclear embryo sac without obvious vacuolation;at the mature embryo sac stage,no embryo sac with completely normal structure and morphology was observed,and the volume of embryo sac of mutant was smaller than that of wild type embryo sac,showing a typical Polygonum type structure,but the degree of vacuolation was low,and the cytoplasmic part was partial There are three kinds of abnormalities in the embryo sac:about 50%of mature embryo sac has complete internal structure,including oophore,two polar nuclei and many antipodal cells,and the position is normal;about 25%of embryo sac lacks oophore,only polar nucleus and antipodal cells can be seen in the embryo sac;about 25%of embryo sac only has many antipodal cells Loss of cell,polar nucleus and oogonium.The proportion of embryo sac with oophore was similar to that of the mutant,indicating that the plants with abnormal embryo sac could bear fruit.4:By using BSA analysis,we sequenced and compared the extreme individuals in BC₁F₄ and BC₁F₅ backcross lines of the semi sterile mutant m21,and obtained the candidate gene sites with SNP and indel differences.Combined with the cytological observation results,we obtained 20 candidate genes that may affect the fertility of female gametes.According to the gene prediction function and gene expression in female gametes,we selected 5 candidate genes?Os01g0102400,os10g0546900,os04g035000,os01g0770400,os06g0638000?.This study laid a foundation for the further study of the fine location and function of the mutant m21.The appearance quality of rice is the main factor that affects the market value of rice,and it also has a certain influence on the yield of rice.It is an important direction of breeding research to breed rice with good appearance quality.Therefore,the research on the appearance quality of rice has always been of great significance.In this study,we used japonica rice"Reyan NO.2"and indica rice"Gui 9b"to construct a recombinant inbred population with a family number of 100,which was successively planted in Guiyang and Sanya.The quantitative genetic trait loci?QTLs?of rice appearance quality in different environments were detected by using the constructed molecular genetic map.The main results are as follows:1:The correlation analysis showed that the correlation between grain length and grain width,grain thickness,chalkiness rate was small,and there was significant positive correlation between grain width,grain thickness,chalkiness rate and appearance quality characters except grain length.This indicated that the regulation mechanism of grain length was different from that of other appearance quality.2:A total of 16 QTLs were identified by QTL detection,which were distributed on chromosome 1,2,3,5,9,10 and 11.The LOD value was between 2.50 and 10.98,and the contribution rate was between 7.64%and 19.89%.3:Two QTLs of grain length were stably inherited.LOD was between 2.62 and 5.06,and the contribution rate was between 7.64%and19.26%.Eight QTLs have not been reported.The contribution rate is between 8.06%and 15.13%.It may be a new QTL of appearance quality,which is worth further study.