Map-based Cloning And Functional Analysis Of The OsMFS2 Gene For Female And Male Abortion In Rice

Rice is one of the most important food crops in the world.With the rapid development of the world today,the number of people is constantly increasing,and the area of arable land is decreasing day by day.Ensuring national food security is an eternal issue.Rice yield is mainly composed of effective panicle number,seed setting rate and thousand-grain weight.Rice seed setting rate is directly related to fertility.Therefore,research on rice fertility is very important to increase rice yield.Meiosis is an extremely special and important step in the process of sexual reproduction in plants.Discovering more genes related to meiosis and analyzing their molecular functions will not only have important theoretical value for studying the molecular mechanism of meiosis,but also provide important genetic resources to improve rice yield.The rice sterile mutant used in this study was selected after mutagenesis by EMS.The F₂ segregating population was obtained by constructing heterozygous plants and indica rice variety N22 hybridization and selfing.With map-based cloning used by the completely sterile extreme individuals and the whole genome resequencing analysis,we located the target gene on chromosome 6.This gene is involved in the pairing and synapsis of homologous chromosomes in meiosis,and the related research has not been reported in rice.We conducted detailed cytological observations on male and female gametes of the mutant,and detected the homologous chromosome pairing and synapsis of the mutant during meiosis,and performed experiments such as gene expression pattern analysis,subcellular localization,and yeast two hybridization.The specific results are as follows:1.Observe and compare the phenotype and fertility of Osmfs2（male and female sterility2）mutant and wild-type plant.In the vegetative growth stage,there was no significant difference between the Osmfs2 mutant and the wild type.However,after entering the reproductive growth stage,the Osmfs2 mutant showed a phenotype that the anthers became whiter and smaller than the wild type,and the spikelets were completely sterile.I₂-KI staining solution was used to check the pollen development.The results showed that the wild-type mature pollen can form pollen grains of uniform size and dark brown normal staining,while the mature pollen of the Osmfs2 mutant had different sizes,shrunken pollen,and cannot be dyeing.The pollen of different developmental stages was observed with acetic magenta dye solution.The pollen of Osmfs2 mutant began dividing unevenly at the dyad stage,and finally pollen of different sizes and no starch filling appeared in the mature stage.Further semi-thin sections were used to observe the development of male gametes.The Osmfs2 mutant was abnormal at the S9 stage of anther development,and the middle layer of the pollen wall was not degraded and remained until the mature stage.The final microspores had no starch filling and irregular shape.DAPI staining was used to observe the chromosomal behavior during meiosis.The Osmfs2 mutant could not form bivalents in the terminal metamorphosis stage,and 24 monovalents could be clearly observed.In order to further explore the fertility of embryo sacs,the embryo sacs at different developmental stages were observed.The results showed that the embryo sac of Osmfs2 mutant began to be abnormal from the functional megaspore stage,and the functional megaspores degraded during the development process,and finally formed cavity embryos.Finally,we used wild-type pollen to saturate the stigma of Osmfs2 mutant and wild-type,and the results showed that the wild-type can produce normal fruit,while the Osmfs2 mutant can not produce fruit and still be completely sterile.These results indicate that both male and female gametes of the Osmfs2 mutant are sterile.2.We found 97 fertile single plants and 37 sterile single plants（97:37,χ²=0.358,p>0.05）by constructing a mapping population of OsMFS2 heterozygous/N22 and performing fertility statistics on its F₂ population The segregation ratio of 3:1 indicated that the mutant phenotype was caused by a single recessive nuclear gene mutation.First,select 10 sterile single plants for initial mapping,and locate the gene on chromosome 6.Subsequently,the population was expanded,and the 141 sterile strains in the population were used to locate the gene within3.4Mb between the chromosome 6 markers MF-14 and RM6818.Because the target gene is located near the centromere,there are a small number of exchange recombined individual plants,and it is difficult to further narrow the localization interval by using traditional localization methods.In this study,the Mut Map+method was used to select 30 phenotypes of fertile and sterile individual plants,extract DNA from each individual plant,and mix the same amount of DNA from each individual plant to construct two pools of fertile and sterile.The two pools were sequenced and the delta SNP index was calculated.Finally,the target gene LOC＿Os06g27860 was determined by combining the sequencing results and the positioning interval.After sequencing,there was a mutation from G to A at the junction of the fourth exon and intron of the gene in the mutant,which resulted in a change in its shearing mode and ultimately a loss of gene function.LOC＿Os06g27860 is annotated as PHS1（POOR HOMOLOGOUS SYNAPSIS 1）in the National Rice Data Center,which is related to the association of homologous chromosomes during meiosis,so this gene is used as a candidate gene.In order to verify the correctness of the candidate genes,we used CRISPR/Cas9technology to knock out the candidate genes at different sites,and obtained two homozygous positive plants Osmfs2-1 and Osmfs2-2 with mutations at different sites.The phenotype and meiotic chromosome behavior of these two knockout plants are consistent with those of the Osmfs2 mutant,indicating that LOC＿Os06g27860 is the target gene OsMFS2.3.In order to further study the function of OsMFS2 gene in rice,firstly,quantitative analysis of OsMFS2 expression in various wild-type tissues showed that the gene was constitutively expressed,but the expression amount is the highest in young panicles.The subsequent quantitative analysis of anthers at different developmental stages showed that the expression of OsMFS2 reached its peak at the S7 stage of anther development.These results were also consistent with the mutant phenotype.Subsequently,through subcellular localization analysis in rice protoplasts and tobacco mesophyll cells,it was found that OsMFS2 was localized in the cytoplasm.However,the expression analysis of meiosis-related genes shows that the loss of OsMFS2 had no significant effect on the expression of these genes.Yeast two-hybrid experiment results also showed that OsMFS2 does not interact with OsRAD50 and OsMRE11.At present,it is not clear how OsMFS2 functions in rice,so the mechanism needs further studies.