Map-based Cloning Of Two Genic Male Sterility Genes In Rice (Oryza Sativa L.)

The anther cuticle and wax layer act as structural support and protective barriers for the development of pollen sac in rice.In this study,we analysis the phenotype and gene mapping on two no-pollen genic male sterility mutant whf41 and whf34 to provide a basis for further cloning and understanding on the molecular mechanism of these two genic male-sterile genes in regulating pollen development in rice.1.The no-pollen male sterility mutant whf41 exhibited thin and transparently milky anther without mature pollen grains,from the mutant progeny of ⁶⁰Co-?-treated indica cultivar Zhonghui 8015?Zh8015?.Compared with wild type,transverse section analysis of the anther development progress showed that the whf41 mutant displayed aborted pollen grains,none detectable exine and swollen tapetal layer,following with no-occurrence of expected PCD?Programmed Cell Death?,finally resulting in fragments of tapetal layer and pollen grains in the chamber.Scanning electron microscopy analysis further revealed the mutant showed smoother anther wall,fewer lipids both on the inner and outer side and fregments of degragated pollen.Genetic analysis revealed that whf41 was controlled by a single recessive genic gene,which was fine-mapped to a 45.6-kb region on the short arm of chromosome 3between markers XD-5 and XD-11,which harboring nine ORFs.Seqencing analysis revealed that the whf41 mutant carried a nucleotide substitution and three nucleotide deletion,which leaded a substitution of one amino acid?D to M?and a deletion of one amino acid?V?in the fourth exon of one Cytochrome P450 Family gene,LOC_Os03g07250,which is named as OsWHF41 and probably responsible for the no-pollen male sterility phenotype.Furthermore,qPCR analysis showed that the expression level of CYP704B2 and seven anther lipid synthesis and transportation related regulators changed significantly in whf41.These results indicated that OsWHF41 played an important role in anther lipid synthesis and pollen exine formation2.The whf34 mutant was aslo isolated from the mutant library of ⁶⁰Co-?-treated indica cultivar Zhonghui8015?Zh8015?indicated thinness and white anthers without mature pollen grains.Transverse section analysis of the anther development showed that the whf34 mutant displayed none detectable exine,degradation of tapetum was pushed back,pollen cell debris mixed with tapetum cells and then these mixture was degradated in the subsequent stages,the anther is in a cavity and did not crack.The SEM results further proved that the whf34 mutant anther wall is smooth and shaped,lack of sporopollenin and ubischbodies,no pollen cells were found.Genetic analysis revealed that whf34 was controlled by a single recessive genic gene,which was fine-mapped to a 57.2-kb region on the short arm of chromosome 7 between markers w46 and w18,which harboring 7 ORFs.Sequence analysis revealed that the first exon of the ORF4?LOC_Os07g42970?carried a nucleotide substitution?A to C?,which resulted in a replacement of a aspartic acid by serine?codon 63?.Furthermore,qPCR analysis showed that the expression level of gene which related to the degradation of tapetum and pollen wall formation were significantly different in whf34,except WDA1.These results indicated that OsWHF34 played an important role in the degradation of tapetum and pollen wall formation.