Phenotypic Characterization And Genetic Mapping Of A New Gene Required For Male And Female Gametophyte Development In Rice

Rice(Oryza sativa) is one of the most important food crops in the word.The rice genome sequence has been determined and a large number of mutants are available. Meanwhile,because of its smaller genome size(430Mb),so rice is an excellent model plant for cereal developmental biology.Male and female sterility phenomenon occasionally occurring in plant,which is bad for the plant themselves but it offer a good chance for our understanding the mechanism of plant development.In rice anther development process,the microspore mother cell firstly undergoes meiosis to produce the microspore in the anther locule,and the microspore further developed into pollens through mitosis,then the anther splited and released maturity pollens for fertilization. Any factors participated abnormally in anther development would resulted in anther abnormality,which finally caused male sterility.The megaspore mother cell undergoes meiosis to form four haploid megaspores.The three of spores undergo programmed cell death and a chalazal spore becomes a functional megaspore.The megaspore then undergoes three sequential mitotic nuclear divisions,to generate the eight nuclei of the mature embryo sac.In the mature embryo sac,the micropylar end of the ovule has the egg cell and two supporting cells called synergids,while the chalazal end of the ovule has three cells of undetermined function called the antipodals that disintegrate prior to fertilization.In recent years,with the development of the rice genome sequencing and annotation,the molecular mechanisms of pollen development in rice are gradually clear.We reported the male and female sterile mutant,MFS-Z9,was generated from a T2 generation transgenic line of normal Japonica rice,Zhonghua 9.The sterility phenotype was subsequently confirmed not be the causation of the foreign T-DNA insertion by linkage analysis.The results of the mendelian genetics,genetic effect, morphological cytology and chromosome localization were as follows:1.With normal flowering,the mutant set no seeds when matured.Also no seeds are obtained neither the mutant is crossed as a pollen receiver nor as a pollen donor. The floret of the mutant consisting of 6 stamens and 1 pistil,looks the same as that of the wild type except that the filaments are long and thin and the anthers are withered in white transparence.The mutant could not produce any fertile seeds except some fake-enlarged ovules when it was crossed with several normal rice lines as pollen receivers.It is verified that in MFS-Z9 no pollen grains can be stained with 1%I₂-KI solution and the anther locules are always hollow.2.Anther transverse section indicates that microspores abnormally degenerated at the stage of meiotic,while the tapetum layer couldn't normally degenerate and remained all the time.To gain a more detailed understanding of the abnormalities of the sporogenous and the tapetal cell,the meiosis stage was divided into several substages for observations according to the state of synapsis and condensation.No obvious differences were observed between the mutant and the wild-type rice during the early premeiosis stage.Morphological differences began at the stage of leptotene in the anther locules.During this stage,the microspores began to degenerate and the microspores were almost completely collapsed at the tetrad stage,while the tapetum layer couldn't normally degenerate and remained all the time.3.In addition,more than 95%of the mutant embryo sac did not undergo differentiation and lacked visible nucellus cells in the ovule,and almost 5%of the embryo sac failed to develop into functional embryo sac with eight cells.Thus,mfs-Z9 displayed multiplex defects in the process of male-female organ development.4.Due to its serious sterility in both male and female organs,the mutation was maintained by heterozygotes(+/-,designated as ZHS,in Zhonghua 9 background). ZHS(+/-) was first crossed with normal rice lines(+/+),then all the generated F₁ plants(+/+ or +/-) were harvested separately,and approximately 100 seeds from each F₁ plant were sown to generate small F₂ populations to investigate the segregation of mutant phenotype.The segregation exactly indicated that the corresponding F₁ plant was in a heterozygote genotype(+/-).All seeds harvested from the selected heterozygote genotypic F₁ plants(+/-) were then sown to produce large F₂ populations for further genetic analysis and mapping.According to this protocol,3 F₂ populations were generated from the cross of ZHS/G630,ZHS/Nipponbare and ZHS/Kitake, respectively.After flowering,all individual plants were subjected to fertility investigations and data were recorded.All F₁ plants exhibited the wild type phenotype, suggesting that the mutant trait was recessive.In the F₂ populations,the segregation of fertile plants and sterile plants fit a ratio of 3:1(X²ï¼œX²_0.05,1=3.84),which indicated that the mutant phenotype was controlled by a single recessive nuclear gene. Considering the mutant phenotype and its defectives,the mutant gene was termed Multiplex Defective Male-female Sterility 1(MDMFS1).5.Polymorphisms between MFS-Z9 and other rice lines,G630,Kitake and Nipponbare were examined,respectively,with 512 pairs of SSR primers.The polymorphic markers were subsequently used to survey in a small population including the 2 parents,4 wild type F₂ plants and 6 mutant F₂ plants.Results showed that 4 SSR markers RM555,RM3732,RM3497 and RM7215,located on chromosome 2,were obviously associated to the MFS-Z9 phenotype.Those 4 markers were then utilized to survey all 226 mutant plants which come from the ZHS×G630 in the same F₂ population.Thus,RM555 and RM3732 were verified to be linked to MDMFS1 on one side,with genetic distances of 1.11 and 0.44 cM,respectively,and the other two markers RM3497,RM7215 were verified to be linked to MDMFS1 on the other side, with genetic distances of 0.44 and 0.89 cM,respectively.The same approach for linkage analysis was employed in another population,ZHS×Kitake which contains 556 mutant plants.Another 2 polymorphic markers,RM3865 and RM12585,with 4 and 1 recombinants revealed,were confirmed to be linked to the MFS-Z9 phenotype,with genetic distances of 0.36 and 0.09 cM on the same side with RM555 and RM3732, respectively.6.To further narrow down the genomic region containing the MDMFS1 locus, more SSRs and InDels(Insertions and Deletions) markers between RM12585 and RM3497 were developed.Out of 80 newly developed markers,4 makers designated as DI014,DI016,DI017 and D051 were polymorphic in the population of G630×ZHS. Linkage analysis showed that marker D051 was tightly linked to MDMFS1 with genetic distance of 0.22 cM,and marker DI014,DI016 and DI017 were all co-segregated with MDMFS1.These results,together with the rice genome sequence data,suggested that the MDMFS1 locus was located on a 99-kb genomic fragment bordered by marker RM12585 and D051 on a single BAC clone on chromosome 2.7.In the 99-kb genomic interval of Nipponbare genome,totally 11 putative genes were predicted by TIGR RICE(http://rice.plantbiology.msu.edu/).Out of them 2 genes are hypothetical protein(LOC_Os02g08430 and LOC_Os02g08460),1 gene is conserved hypothetical protein(LOC_Os02g08470) and 1 gene is transposon protein (LOC_Os02g08450);while the remaining 7 genes are expressed proteins. LOC_Os02g08440 is a member of the Superfamily of rice TFs having WRKY and zinc finger domains;LOC_Os02g08480 is ubiquitin fusion degradation protein 1; LOC_Os02g08490 is chaperone clpB 1;LOC_Os02g08500 is the two-component response regulator ARR12;LOC_Os02g08510 is zinc finger protein 1; LOC_Os02g08520 is proteasome subunit beta type 3 and the LOC_Os02g08530 is a phytosulfokine receptor precursor.