Cytological Observation And Genetic Diversity Analysis Of Newly Selected CMS Materials And Subgroups Of CMS-C Materials In Maize

Cytoplasmic male sterility(CMS)plays a crucial role in the hybrid seed production of heterosis utilization in maize.However,planting single sterile cytoplasm in long term with large acreage will lead to the potential risk of the outbreak of dominant physiological races, such as maize CMS-T used in American during the central period of 20 century.There for, it is very important to create and excavate new types of maize CMS material and increase the genetic basis diversity of male sterile material.In the present study,9 newly selected CMS materials together with 3 subgroups of CMS-C type had been studied.PCR were conducted with 3 pairs of specific primers corresponding to maize CMS-T,CMS-C and CMS-S, the cytological characteristics of anther abortion and cell apoptosis during anther development were observed,genetic diversity at mtDNA level were analyzed by AFLP. Besides,the tested materials were inoculated and identified with Bipolaris maydis Race C The purpose of this study was to identify the newly selected cytoplasmic male sterile materials with different sources and seek a simple and effective method for genetic diversity detection.The main results were as followings:1.The tassel fertility identification of the 9 newly selected CMS materials in the background of 18 red and RP125 were investigated,all the spikelet were thin and small, without any anthers exserted. Moreover,microscopic examination showed that there were no pollen grains in the anthers for some materials and others with pollen grains but can not be stained.2.PCR analysis were carried out with the newly selected CMS materials by the specific primers CMS-T,CMS-C,CMS-S in maize,the TMo17,CMo17 and SMo17 were used as checks.The electrophoretic results suggested that SauS1,SauS2,SauS3,SauW1,SauW2, SauA1,SauA2 and CMol7 can amplify a same size fragment,furthermore,SauS4,SauS5 and TMo17 amplified another same size fragmentboth in the backgrounds of 18 red and RP125.These results manifested that SauS1,SauS2.SauS3,SauW1,SauW2,SauA1,SauA2 might belong to the C type male sterile cytoplasmic,and SauS4 and SauS5 might belong to the T type male sterile cytoplasmic.3.Paraffin section and TUNEL technique were carried out for the observation of pollen development with newly selected CMS materials and CMS-C type subgroup materials. Major results were as follows:?The microspore abortion of SauSl,SauS2, SauS3,SauW1,SauW2,SauA1 and SauA2 mainly occurred from the tetrad stage to the middle of mononuclear stage,and their tapetum behaved delayed degradation.Thus.these materials showed a similar abortion characteristics with CMS-C type in maize. In contrast, the microspore abortion of SauS4 and SauS5 mainly occured from the middle of mononuclear stage to the later of mononuclear stage, the tapetum cells degraded ahead of the time.SauS4 and SauS5 showed the similar abortion characteristics with CMS-T type.? Cytological observations with 2 nuclear backgrounds of 48-2 and Huangzaosi suggested that the microspore abortion of the C cytoplasm (CI subgroup) was mainly occurred in the period of tetrad to the middle of the mononuclear stage.Meanwhile,periclinal division were observed in part of the tapetum cells.In the RB cytoplasm (CII subgroup),the microspore abortion mainly occurred from the early stage of mononuclear till the middle stage of mononuclear.The microspore abortion of ES cytoplasm (CIII subgroup) primarily occurred from the tetrad stage to the middle stage of mononuclear period. ?Through the observations of the microspore development process of the 9 newly selected male sterile cytoplasms under 18 red and RP125 backgrounds and subgroups of CMS-C type with the background of Huangzaosi and 48-2,different abortion feature were observed under the different nuclear background,indicated that not only the cytoplasm but also the nuclear background have influence on the abortion style of CMS lines.4.AFLP technique was used to analyze the mtDNA diversity of the newly selected CMS materials under 18 red and RP125 nuclear backgrounds and CMS-C subgroup materials with the nuclear backgrounds of 48-2 and Huangzaosi.?8 primer pairs of total 64 primers exhibited polymorphism including E31M31,E32M35,E33M33,E34M37,E37M31, E37M33,E37M35,E37M36.Approximately 15 bands were amplified by each pair of primer in one material.?The amplification results of the primer E33M33 showed that SauS4 and SauS4 in the background of 18 red and RP125 were significantly different from those of SauS1,SauS2,SauS3,SauW1,SauW2,SauAl and SauA2.We concluded that SauS5,SauS4 might belong to different cytoplasmic types with the other 7 CMS materials. At the same time,it was found that different PCR products between SauW1 and SauW2,SauA1 and SauA2 were also detected.Although the amplified bands of C?,C?,C? existed some difference by the primer E33M33,but this primer could not yet to distinguish the 3 subgroups of C?,C?,C? from each other.This result further indicated that it was probable to distinguish the different subgroups of CMS-C from DNA level.5. two nuclear backgrounds of 9 newly CMS materials were inoculated with Bipolaris maydis Race C for two different planting periods,and Mo17,TMo17, CMo17, SMo17 were tested as checks.The results showed that the disease condition of CMo17 was higher than those of TMo17,Mo17 and SMo17.And the disease indexs of SauS1,SauS2,SauS3, SauW1,SauW2,SauA1 and SauA2 were higher than that of CMo17.The disease index of SauS4 and SauS5 were lower than that of CMo17.The results showed that the Bipolaris maydis Race C used in this study had the specific infection ability to CMS-C in mazie. From this experiment we could find the disease index of CMS lines with the 18 red background were higher than that of RP125.The results indicated that the resistance to Bipolaris maydis C race of CMS cytoplasmic varywith different nuclear backgrounds of CMS lines.