Cytological Observation And Transcriptional Analysis Of Thermo-sensitive Genic Male Sterile Line SP2S In Brassica Napus L.

The thermo-sensitive genic male sterility(TGMS)line SP2 S is a spontaneous rapeseed mutation,and it is a kind of sterile type which is of practical value in the breeding of two-line hybrids.To uncover the key cellular events and genetic regulation,and provide a foundation for deciphering the transcriptional regulation of SP2 S in response to temperature change associated with TGMS expression,a combined study using cytological observation,transcriptome profiling,gene expression real-time PCR and de novo transcriptome reconstruction was conducted for SP2 S and its near-isogenic line SP2 F grown under warm conditions.The results suggested:1.Asynchronous microsporocyte meiosis,abnormal tapetal plastids and elaioplasts were demonstrated in the anther of SP2 S.The tetrad microspore did not undergo mitosis before the cytoplasm degenerated.Delayed degradation of the tetrad wall,which led to tetrad microspore aggregation,resulted in postponement of sexine(outer layer of pollen exine)formation and sexine fusion in the tetrad.The nexine(foot layer of exine)was also absent.The delay of tetrad wall degradation and abnormality of the exine structure suggested that the defective tapetum lost important functions.2.Four groups of mRNAs derived from young floral buds of SP2 S and SP2 F,both of which were cultivated under cool and warm conditions,were sequenced on an Illumina Solexa platform,producing 239.7 million short reads.The length of the short reads were19.95 Gbp.Uing the The reads were assembled de novo using the Trinity program,which resulted in 135,702 transcripts with an average length of 784 bp,an N50 value of 1221 bp and a total length of 107 Mbp.The sequences of transcripts have been deposited in the NCBI Transcriptome Shotgun Assembly(TSA)database(accession GDFQ00000000)24,157cDNA-derived simple sequence repeats were identified in the assembly.When comparing sample SP2 S at 22°C vs.SP2 S at 16°C and sample SP2 S at 22°C vs.SP2 F at 22°C,we found137 and 195 single-nucleotide polymorphisms and 49 and 51 differentially regulated KEGG orthology groups,respectively.The various differentially expressed genes and the derivedsingle-nucleotide polymorphisms showed abnormal transcriptional regulation in the TGMS system.These results outlined a tanglesome transcriptional regulation that occurred in the rapeseed TGMS SP2 S when the temperature changed.About 86.9% of the total transcriptome sequences matched the Brassica database in NCBI,but most of them were unknown at present,so the information of the transcriptional sequences was a useful supplement to the gene and function annotations in NCBI.It also provided an important reference for gene recognition in several transcriptional sequencing schemes of rape(Brassica napus).3.The comparison of transcriptome differences was done using DGE,but its gene sequence was located or compared to the whole transcriptome spliced.A total of 465 differentially expressed transcripts(DETs)were identified based on transcriptomic comparisons between young flower buds of SP2 S and SP2 F plants,including 303up-regulated DETs and 162 down-regulated DETs in SP2 S.Several genes encoding small RNA degrading nuclease 2,regulatory subunit A alpha isoform of serine/threonine-protein phosphatase 2A,small RNA 2′-O-methyltransferase,transcription factor bHLH25,thioredoxin reductase 2,leucine-rich repeat receptor kinase At3g14840 like,glycine rich protein 1A and fasciclin-like arabinogalactan proteins FLA19 and FLA20 were greatly depressed in SP2 S.Interestingly,a POLLENLESS3-LIKE 2 gene encoding the Arabidopsis MS5 homologous protein,which is necessary for microsporocyte meiosis,was down-regulated in SP2 S.Other genes encoding glucanase A6,ethylene-responsive transcription factor 1A-like,pollen-specific SF3,stress-associated endoplasmic reticulum protein 2,WRKY transcription factors and pentatricopeptide repeat(PPR)protein At1g07590 were up-regulated in SP2 S.The tapetum-development-related genes,including BnEMS1,BnDYT1,and BnAMS,were slightly up-regulated in 3-mm-long flower buds or their anthers,and their downstream genes,BnMS1 and BnMYB80,which affected callose dissolution and exine formation,were greatly up-regulated in SP2 S.This aberrant genetic regulation corresponded well with the cytological abnormalities.The results suggested that expression of TGMS associates with complex transcriptional regulation.Based on these evidences,a simple working model of TGMS was established.