Transcriptomic Study On Pollen Abortion Of CMS Pepper(Capsium Annuum L.)

Pepper is a very important vegetable crop with significant heterosis.One important way is the use of CMS Pepper conduct its genetic improvement.The CMS line 8214A is an inability to produce functional pollen,and has already been extensively utilized for hybrid pepper production,8214A is an excellent CMS line pepper with 100%of sterility and superior economic characteristics.However,its mechanisms of CMS pepper at the molecular level remain largely unclear.In this study,the differences in the anther development of Pepper CMS line 8214A and its corresponding maintainer line 8214B were studied by cytology.Microscopic observation of tissue section showed that CMS line 8214A was completely aborted after the meiosis.To clarify the time and cause of pollen abortion,difference on the microsporogenesis and tapetum development in the anthers of male fertile maintainer line and CMS line pepper were studied using transmission electron microscopyThe results showed that,as early as prophase I,some MMCs in CMS line pepper appeared aberrant chromatin migration and formed micronuclei,and vacuolization in tapetal cells And then,MMCs in the same anther locus developed asynchronously,even small part of MMCs degenerated in advance,which appeared the typical morphological hallmarks of PCD,such as condensed and marginalized chromatin in the nucleus,shrunk cytoplasm and vacuolated organelles,And most MMCs could complete the meiosis,but formed non-tetrahedral tetrad microspores with irregular shape and different size and uncertain number of nuclei,The tapetal cells in CMS anthers occurred to the premature PCD after prophase?,finally all aberrant tetrad and tapetal cells degenerated in situ and could not continue to develop the formation of functional pollen.Therefore,pollen abortion in CMS line anthers happened by PCD themselves,and the premature PCD of tapetal cells were closely associated with male sterility.Obviously,the pepper CMS line 8214A pollen abortion is different from most of the CMS due to the abnormalities of the tapetal cells.In order to further reveal the basic molecular mechanism of pepper CMS line 8214A pollen abortion,We used high-throughput sequencing technology Illumina HiSeq^TM PE150 to compare the gene expression of pepper CMS line 8214A and male fertile maintainer line 8214B anthers meiosis,The results showed that 8214A and 8214B were respectively clean reads162107002 and 151227618,and predicted 6165 new transcripts.To identify putative differentially expressed genes?DEGs?between 8214A and 8214B anthers during meiosis,the|log₂ Ratio?S vs F?|?0.6 and Pval<0.05 were used as the threshold in judging the significance of a gene expression difference.1355 genes were found to be differentially expressed,among which 424 genes were significantly up-regulated and931 down-regulated in the CMS anthers compared to those of the fertile.20 DEGs?11 down-regulated genes and 9 up-regulated genes?from our sequencing results were selected and detected by quantitative real time RT-PCR?qRT-PCR?assays.,all of the 20 DEGs showed the same expression patterns in the qRT-PCR assays as in the RNA-seq data.And there was a strong positive correlation?R²=0.9587?between RNA-seq data and qRT-PCR data.Therefore,the RNA-Seq data were highly credible.GO enrichment analysis of the differentially expressed genes showed that the ubiquitin ligase and cell cycle related genes were apparently restrained in the CMS anthers,while methytransferase genes were activated.These genes are involved in the process of DNA damage repair during pollen mother cell meiosis and are closely related to cell death and cell cycle regulation.This result is consistent with the results of cytology and is considered to be related to CMS line 8214A pollen abortion PCD-related genes.