| Maize is the largest grain crop in China and one of the first crops to utilize heterosis.Male sterility as a genetic tool,can not only save the cost of traditional artificial emasculation but also guarantee the purity and quality of maize hybrid seeds,improving seed production efficiency.Therefore,it is of great theoretical and practical importance to explore male sterility genes in maize hybrid production.In this study,the male sterile sister cross population Ky335 MS obtained by natural mutation was used as the material.Through morphological comparison and cytological observation,combined with classical genetics and molecular biology techniques,the sterile characteristics and genetic pattern of the mutant Ky335 ms were identified,and the preliminary mapping of sterile gene was performed to lay the foundation for further fine mapping and candidate gene cloning.The main results are as follows:1.Phenotypic observations results: By observing the phenotype of the male sterile mutant Ky335 ms,we found that the vegetative growth phenotype of the mutant Ky335 ms has no difference from that of the fertile plant Ky335 F in the sister cross population.However,the anther of this mutant became small in size and light yellow in color.At the late stage of anther development,the anthers were dry and wrinkled and unable to disperse powder normally.The mutant Ky335 ms anthers could not be stained by anther starch I2-KI staining,which verified that no mature pollen grains were produced in the anther cavity of the mutant,indicating that the mutant Ky335 ms was completely sterile and was a typical "pollenless" type of male sterility.2.Cytological observations results: 1% magenta acetate staining revealed that mutant Ky335 ms formed tetrads with variable morphology and unequal cytoplasmic division.At the microspore stage,the microspore was deformed,and finally disintegrated and disappeared.The results of semi-thin section observation of anthers showed that the mutant can form a complete four-layer anther wall structure,and the microspores could be released from the tetrad.However,at the middle and late stages of microspore development,the microspores gradually shrink and degrade.The tapetum cells degenerated abnormally and vacuolized gradually.At the stage of mature pollen grains,the anther wall collapsed,and there were no pollen grains in the anther chamber.These results indicated that the sterilization of the mutant Ky335 ms occurred at the microspore stage.3.Genetic analysis results: The mutant Ky335 ms has exhibited infertility in different environments,indicating that the sterile trait of the mutant can be stably inherited and is not affected by the environment factors.The phenotypic segregation ratio of the cross progenies of fertile and sterile plants in the sister cross population was 1:1,whereas the phenotypic segregation ratio of the self-progeny of fertile plants from this population was 3:1,suggesting that the sterile phenotype of the mutant was controlled by a recessive gene.The reciprocal populations were constructed with the mutant Ky335 ms and eight normal inbred lines,in which all F1 generations were fertile,and the fertile segregation ratios of F2 and BC1 were 3:1 and 1:1,respectively.Overall,it indicates that the sterile trait of the mutant Ky335 ms is controlled by a recessive nuclear gene.4.Sterility gene mapping: The(Ky335ms×K115)F2 population was used as the mapping population,and the candidate gene was mapped by the SSR-BSA method.First,a total of 179 pairs of markers with polymorphism were identified from 567 pairs of SSR markers using the two parents.Then,five pairs of markers with polymorphism were identified using the F2 extreme mixed pool.Further,these five pairs of markers were used to genotype and perform linkage analysis on 319 recessive sterile plants in the F2 population.Finally,the candidate sterility gene ms335 was preliminarily located on the long arm of chromosome 9,within the 1.27 c M range between the SSR molecular markers bnlg430 and umc2339.The research results laid the foundation for subsequent fine mapping and sterility gene cloning. |
