Cytoplasmic Effect Analysis And Study On Sterility Mechanism Of Tobacco Male Sterile Line

Cytoplasmic male sterility?CMS?lines and hybrid varieties used in tobacco production in China accounts for more than 70%of the total planting area of flue-cured tobacco,but its sterile cytoplasm all comes from N.suaveolens,which is a type of sua-CMS.sua-CMS is the only sterile cytoplasm identified as having no negetative effect on agronomic traits and resistance of tobacoo,but the mechanism of its abortion is unclear.In order to make better use of this CMS type,it is necessary to dig out the CMS genes related to abortion and the formation mechanism of abortion.In addition,due to the long-term use of a single sterile cytoplasm?sua-CMS?in tobacco breeding and production,the genetic basis is fragile and has great risks.It is urgent to create and identify new type of CMS.And it is necessary to evaluate the agronomic traits and disease resistance of existing sterility types.In this study,a new type of CMS?tab1-CMS?was first identified,and agronomic traits,disease resistance investigation were performed on seven existing tobacco CMS at two points a year.And the cytoplasmic effects of these 7 sterile cytoplasms were analyzed.The floral buds of sua-CMS line and its isonuclear alloplasmic line has been sequenced for transcriptome analysis to study the molecular mechanism of its abortion.The main research results are as follows:?1?Morphological and cytological observations were used to determine that tab1-CMS tobacco abortion occurred at the early anther development stage of sporogenous cells.Under the scanning elelctron microscope,the pollen grains of tab1-CMS were shrunken and their surface had deep grooves.The pollen grain vigor experiment showed that its nonviable.The result of the abortion rate survey showed that it was completely aborted.By sequencing of the trnC-trnD intergenic region of chloroplast DNA of tab1-CMS,sua-CMS,and fertile tobacco,25 SNPs and InDels were obtained,using a specific primer pair based on these polymorphic sites,a 588 bp fragments was amplified specifically in tab1-CMS.?2?Compared the agronomic traits and disease resistance of 7 CMS lines?sua-CMS,glu-CMS,rep-CMS,rus-CMS,tab1-CMS,tab2-CMS,tab3-CMS?and fertile tobacco,we found that there was no significant difference in agronomic traits between sua-CMS,tab1-CMS,tab3-CMS and fertile tobacoo,environment is the main factor affecting the difference of agronomic traits between tab1-CMS and fertile tobacoo.tab3-CMS is less resistant to bacterial wilt than fertile tobacco,but they still belong to the same disease degree,and other CMS lines showed no significant difference in disease resistance with fertile tobaccos,and tab1-CMS tobacco was superior to fertile tobacco in its resistance to CMV and black shank.?3?MSZY100 and ZY100 were used as materials,using morphological and cytological observations to determine that the sua-CMS tobacco abortive period occurs before the differentiation of sporogenous stage and corresponds to a flower bud of?2 mm size.Select this size of MSZY100 and ZY100 flower bud for transcriptome sequencing,get 462 differentially expressed genes?DEGs??p-value adjusted<0.001?.16 DEGs were selected for qRT-PCR verification,the qRT-PCR reults were consistent with the transcriptome results,proving that the transcriptome sequencing results were reliable.?4?The GO and KEGG annotation analysis of the DEGs revealed that these genes mainly enriched in the heat shock protein family,protein processing in the endoplasmic reticulum,proton exchange coupled with energy synthesis,and cell morphogenesis.Further analysis of the DEGs revealed that the synthetic genes of the F₁F₀-ATPase subunit in mitochondria were down-regulated,by measuring the ATP content of the MSZY100 and ZY100 small flower buds,it was found that the internal ATP content of the MSZY100 is only 52%of the ZY100.ERQC,ERAD,and UPR genes of the endoplasmic reticulum were down-regulated in MSZY100 anther cells and PCD related genes were up-regulated.Abnormal PCD appeared in MSZY100 anther cells by observing the microstructure of anther cells using transmission electron microscopy.We speculated that ER stress caused by misfolded proteins leads to PCD and deficiency of ATP synthesis eventually lead to abortion in sua-CMS tobacco.