Identification Of Sterile Gene And Its Abortion Mechanism Analysis Of Nsa CMS In Brassica Napus L.

Cytoplasmic male sterility,as one of the main ways of heterosis utilization,plays an important role in agricultural production.As the type of cytoplasmic male sterility in rapeseed production is single,it is urgent to increase the yield of oilseeds and solve the problem of singularity of cytoplasm utilization.Nsa CMS,as a new type of allo-cytoplasmic male sterility line with independent intellectual property rights in China,is different from the current Nap CMS,Ogu CMS,Pol CMS,Tour CMS,etc,and was completely stable abortion.At present,three-line combination for the Nsa CMS system has been realized.It is expected to select excellent hybrid combinations by this system with great potential for breeding applications.However,the corresponding sterility gene and abortion mechanism for this sterility system are still unknown.Based on the previous study,Nsa CMS and maintainer line mitochondrial genome were compared to identify the specific open read frames(ORFs)in Nsa CMS.Through further expression analysis,genetic transformation,functional and subcellular localization,we have determined the Nsa CMS-associated gene and its function.Through the cytological observation,reactive oxygen species and energy metabolism,endogenous hormones and transcriptome analysis,we revealed the abortion mechanism of Nsa CMS.The main results obtained in this study are as follows:1.Phenotypic and cytological observation showed that the abortion characteristics of Nsa CMS were different from those of Pol CMS and Ogu CMS,with complete abortion and early anther degeneration.The observation of semi-thin section showed that the tapetum of Nsa CMS pollen cell became thinner from the development stage to the tetrad stage,showing obvious signs of "abortion",and could not form normal microspores in the uni-nuclear stage result in pollen abortion.2.Mitochondrial genome analysis of Nsa CMS showed that its mitochondrial genome mainly originated from Sinapis arvensis.Sequences of 16 functional genes were completely same in the three materials of Nsa CMS,Zhongshuang 4 and Sinapis arvensis.5 and 13 mitochondrial genes were completely same Zhongshuang 4 and S.arvensis,respectively.11 ORFs were identified to be specific and with a transmembrane domain by comparing the mitochondrial genome of sterile line and maintainer line of them which may be related to the sterility of Nsa CMS.3.The analysis of candidate sterile genes showed that the expression of orf346 in restorer lines was significantly inhibited.Northern blot showed that orf346 had specific hybridization bands in sterile lines,and it was confirmed that orf346 was co-transcribed with nad3 and rps12.Further genetic transformation experiments showed that orf346 was the cause of pollen abortion in Nsa CMS.4.The study on the function of orf346 showed that it can inhibit the production of energy and increase of active oxygen in cells.The subcellular localization showed that the sterile protein located in the inner membrane of mitochondria.The analysis of expression of mitochondrial functional gene showed that nad1,nad2,nad5,nad6,cox1,cox2-2,cob F and other genes involved in respiratory chain were significantly or extremely significantly down regulated in the critical S1 period of abortion.The results showed that the expression products of orf346 gene might play a role by inhibiting the expression of some functional genes in mitochondria.5.The results of transcriptome sequencing showed that the different metabolic pathway genes are enriched in starch and sucrose metabolism,energy generation and conversion,active oxygen,hormone signal transduction pathway.In addition,ABA,SA and IAA were found to be different in the male sterile line and maintainer line.The results showed that the abortion of nsacms might be related to hormones,energy and reactive oxygen species.6.According to the sequence of orf346 gene,a pair of specific markers were developed and tested in 24 collected materials.The results showed that only the male sterile line and its corresponding restorer NR1 could amplify the target band of orf346 gene,and no other germplasm resources or varieties could amplify the target band.The marker could identify the materials containing the cytoplasm of Nsa CMS.Based on the current research results,orf346 was integrated into the mitochondrial genome of Brassica napus during the process of somatic recombination.By interfering with the electron transport chain,the expression products of sterile gene caused the changes of cell energy,active oxygen and hormone,and the down-regulation of key genes of pollen development,which eventually lead to the abortion of Nsa CMS.