Molecular Mechanism Of Temperature Sensitivity Of Pol CMS In Brassica Napus L.

Polima cytoplasmic male sterility(Pol CMS)is one of the most important types of male sterility utilized in hybrid breeding in Brassica napus.Yang et al.(1995,1998)identified temperature-sensitive Polima cytoplasmic male sterility(Pol TCMS),i.e,being male fertile at low temperature situation and male sterile at high temperature situation,in the offspring of a cross beween two Brassica napus varieties.On this basis,some pol TCMS AB lines with the characteristics of easily reproduction and high yield F1 seed production were developed,and dozens of elite hybrid varieties,such as ?Huayouza 10?,were registered successively in China.Further study by Yang et al(1997)revealed that multiple quantitative genes in nucleus might regulated temperature sensitivity of Pol TCMS.In order to study the fertility conversion molecular mechanism of pol TCMS,a temperature-sensitive sterile line(Y402)and a stable-fertile restorer(Rf48)of B.napus were treated with 25 °C and 10 °C during the critical period of bud differentiation,and RNAs were extracted from 0.5-mm buds at the key stage of fertility conversion.After transcriptome analysis,a few genes related to Pol TCMS fertility-conversion were identified.Meanwhile,combining with the previous research of pol CMS restoration genes,the molecular mechanism of fertility conversion in Pol TCMS was preliminary discussed.The results are summarized as follows:1.RNA sequencing was performed on Illumina HiSeq-150 platform,and raw data 155.893 G and filtered clean data 154.736 G were obtained.The clean data was mapped to the ZS11 reference genome by Hisat2 and the mapping rate was about 90%.Transcripts were assembled by StringTie to yield 199589 unigenes with a total length of 267 Mbp.2.Differential gene expression analysis was performed using Ballgown and DEseq2.The threshold was set as FDR<0.01 while the differential expression more than 2 times.In Y402,910 differentially expressed genes were obtained,including 439 upregulated genes and 471 downregulated genes;in Rf48,1808 differentially expressed genes were obtained,including 990 upregulated genes and 818 downregulated genes.3.The differentially expressed genes of Y402 were subjected to GO and KEGG enrichment analysis by Blast2 GO.The GO enrichment analysis found that there was an enrichment of downregulated genes focusing on cell composition,anabolism,and reproductive growth,and upregulated genes focusing on energy supply-related functions such as photosynthesis and redox reactions in Y402 under the high temperature environment.The KEGG enrichment analysis results were consistent with the results of the GO enrichment analysis,and the oxidative phosphorylation pathway,containing ofr224/atp6 which is involved with fertility conversion in Pol CMS,was enrichmented.It's worth noting that in this pathway,there is an important gene MSTRG.5508.4 which is silenced in high temperature environment but has high expression level in low temperature environment in this study.It suggested that MSTRG.5508.4 may be a key gene responsible for the fertility conversion in Pol TCMS under high temperature environment.4.Through the double alignment method,genes related to the fertility were screened in the differentially expressed genes in Y402.The results showed that Pol CMS fertility maintenance gene rfp was downregulated at high temperature;transcription factors SPL1 and SPL12 which confer plant thermotolerance at reproductive stage were downregulated at high temperatures;genes ROXY1,MYB65,ER,etc,which are considered to regulate anther development in early meiotic stages,were downregulated at high temperatures.