| Heterosis is one of important way to improve the wheat quality and yield.Physiological male sterility induced by chemical hybridization is one of the effective methods to utilize wheat heterosis.The utilization of SQ-1 avoids the tedious process of stamen removal and male sterile wheat induced by SQ-1 could be used as the parent of hybrids wheat production.Wheat K-type cytoplasm male sterility lines are recognized as one of the most potential male sterile types in utilization of wheat hybridization with the characteristics of stable sterility maintenance,high germination rate and good agronomic traits.Xinong 1376-CIMS treated with SQ-1 and normal Xinong 1376,K-type sterile line ms（Kots）-90-110 and its fertility restoration near isogenic line RK90-110 were used as the experimental materials in this study.The cell morphological characteristics of anthers and microspores at different developmental stages were observed and analyzed by using stereomicroscope and scanning electron microscopy.In order to elucidate the mechanism of SQ-1-induced physiological male sterility,the TaICK1 was used as the target of this research.We preliminarily located the restorer gene of wheat K-type male sterility line ms（Kots）-90-110 by using wheat 660K SNP chip.Transcriptome sequencing analysis was performed between ms（Kots）-90-110 and RK90-110 at the uninucleate stage,the binucleate stage and the trinucleate stage of the anthers,and the sequencing results were verified by q RT-PCR analysis,which provided a basis for elucidating the restoration mechanism of K-type wheat cytoplasmic male sterility.The main results of this experiment are as follows:1.The full-length c DNA sequence of wheat TaICK1 gene was successfully cloned from Xinong1376.Real-time PCR indicated that the expression pattern of TaICK1 in wheat different tissues showed significantly tissue-specific,with the highest expression level in anthers and the lowest expression level in roots.In SQ-1-treated wheat male sterile material Xinong1376-CIMS,the expression level of TaICK1 was significantly higher than that in Xinong1376 at the early uninucleate stage,late uninucleate stage and trinucleate stages of microspore.These results demonstrate for the first time that the SQ-1 could induce the expression of TaICK1 in common wheat.2.In order to verify the function of TaICK1,we have created transgenic rice lines that overexpress the wheat TaICK1 gene.The results showed that overexpression of TaICK1 gene in rice caused a significant decrease in seed setting rate.In addition,we found that overexpression of wheat TaICK1 gene in rice also caused a significant delay in the heading period.To our knowledge,this is the first discovery that the ICK1 gene is related to the heading period.These results indicated that higher levels of TaICK1 may be one of the key factors leading to a decrease in fertility of Xinong1376-CIMS.3.We identify two cyclins protein,Ta CYCD2;1 and Ta CYCD6;1,by using the Yeast two-hybrid system.The expression levels of genes Ta CYCD2;1 and Ta CYCD6;1 in Xinong1376-CIMS were significantly lower than that of Xinong1376.The expression levels of Os CYCD2;1 and Os CYCD6;1 in transgenic rice plants were also significantly lower than those of wild-type.The results suggested that SQ-1 was not related to the decrease of Ta CYCD2;1 and Ta CYCD6;1 expression in Xinong1376-CIMS.We speculate that SQ-1induces an increase of TaICK1 in Xinong1376-CIMS firstly,and then the excessive TaICK1 in cell may inhibit the expression level of Ta CYCD2;1 and Ta CYCD6;1 in Xinong1376-CIMS.These results suggested that SQ-1 induced wheat male sterility is the result of coactions from TaICK1,Ta CYCD2;1 and Ta CYCD6;1.4.The wheat 660k SNP chip was used to scan the genotype of DNA pools constructed from F2population of cross ms（Kots）-90-110×RK90-110.We found that the 74.9%SNPs with polymorphism are mapped on the 2D chromosome of wheat,and the region with the highest distribution density is 571-620Mb of wheat 2D chromosome.Therefore,we speculate that the restorer gene is located in this region,and it may be a new restorer gene.5.Transcriptome sequencing analysis was performed between ms（Kots）-90-110 and RK90-110 at the uninucleate stage,the binucleate stage and the trinucleate stage of the anthers.A total of 45859 differentially expressed genes were found.There were 1409differentially expressed genes at the uninucleate stage,the binucleate stage and the trinucleate stage of the anthers.According to the annotated KEGG metabolic pathways and GO enrichment analysis,we proposed a fertility mechanism centered on energy metabolism.Disturbance of energy metabolism may be the core reason for the abortion of sterility lines,which provides clues for us to further explore the recovery mechanism of sterility.6.In this study,we conducted a joint analysis of the results of gene mapping and the results of the transcriptome at the binucleate stage.Among the 761 highly reliable genes within the candidate intervals on 2D chromosomes,only 248 genes showed expression difference between sterile line and fertile restorer line.Among the 248 differentially expressed genes obtained,48 genes were annotated to specific metabolic pathways,and 5 genes related to energy metabolism of them were identified as candidate genes. |
