| Wheat(Triticum aestivum L.)is one of the most important food crops.The utilization of crop heterosis has played an important role in improving yield,quality,and stress resistance,and has obtained great social and economic benefits.The utilization of wheat heterosis has become one of the important choices.Thermo/photosensitive cytoplasmic male sterile lines play an important role in the utilization of heterosis in wheat.YS3038 is a YS-type thermosensitive male sterile line.During the meiosis period of microspore development to the mononuclear period,when the average temperature is lower than 18~oC,it is completely sterile,which can be used as a sterile line;when the average temperature is higher than 20~oC,it is fully fertile,which can be used as a maintainer line.However,the molecular mechanism of the YS3038 fertile conversion is unclear.The purpose of this study is to explore the fertility conversion mechanism of YS3038.And a systematic study on thermosensitive male sterile line YS3038 was carried out.To determine the key stage of fertility transformation,cytological observations of anther and microspore growth were made and the transformation period of fertility was determined.Gene mapping analysis of the sterile genes was carried out to identify the candidate sterile genes.In order to further screen the candidate genes on mapped interval and other fertility related candidate genes,the transcriptome analysis was performed on YS3038,and the barley stripe mosaic virus-induced gene silencing(BSMV-VIGS)validation was performed on the key genes obtained.To further understand the upstream regulators of candidate genes,the non-coding RNAs of YS3038 were identified and an important fertility conversion mechanism was predicted.The main research results are as follows:1.The development of pistil was completely normal,and the anthers were slender and not dehiscent during flowering,resulting in the sterility for self-cross under the sterile conditions(pollen pattern).Under the sterile conditions,the chloroplasts in the endothecium cells of YS3038 during the meiosis stage were defective.And the tapetum cells were degraded in advance during the meiosis stage,but no obvious elaioplast and tapetosome were observed.Under the sterile conditions,plasmolysis occurred at the late uninucleate stage of the microspores.And at the binucleate stage,the contents of the cytoplasm of the microspores were less and uneven,the staining of microspores was shallow,the vacuolization of microspores was serious,and the microspores were irregular.It proved that the microspores at the binucleate stage were abnormal.The microspore development was hindered from the late mononuclear stage to the binuclear stage via fertility conversion experiments.2.Sterile genes were mapped on chromosomes 1B and 6B by using the extreme individual hybrid pool and 660K chip of wheat.Further analysis mapped the male sterile genes on two regions on chromosome 1B,81 Mb-91 Mb and 363 Mb-389 Mb,and one region on chromosome 6B,480 Mb-520 Mb.The two loci on chromosome 1B contained 283genes.The genetic linkage map of chromosome 1B was constructed,and a major QTL was mapped on 1B,named as Tae TCMS1,with a genetic distance of 7.23 c M,which could explain 27.3190%of the phenotypic variation.The location of this QTL was basically consistent with 81 MB-91 Mb on chromosome 1B,and there were 31 candidate genes in this region.3.The different gene expressions were involved in the anther development of YS3038at different reproductive stages.Under different conditions,the number of differentially expressed genes at the binucleate stage was the largest.Four of the candidate genes on the1B mapping interval were differentially expressed at the binuclear stage,and two of them were annotated to the pathways of lipid transport and metabolism and signal transduction.Besides,weighted gene coexpression network analysis(WGCNA)showed that two modules were highly correlated with anther development and fertility conversion,respectively.Among them,differentially expressed genes in the fertility conversion module were enriched into 16 KEGG pathways related to energy metabolism.The BSMV-VIGS silencing analysis of candidate genes found that the seed setting rates were significantly reduced after the silencing of Leucine-rich repeat receptor-like protein kinase,Tae RPK,and Fatty acyl-Co A reductase 5,Tae Far5.The gene expressions of silenced plants were significantly reduced.Therefore,Tae RPK and Tae Far5 genes are probably involved in the male sterility process of YS3038.4.With the microspore development,the proportions of up-regulated lnc RNAs and circ RNAs under sterile conditions increased accordingly,and the number of differentially expressed mi RNAs also increased accordingly.In the binuclear phase,the target genes with opposite expression patterns of differentially expressed mi RNAs played important roles,which were mainly enriched in the degradation of aromatic compounds,ubiquinone and another terpenoid-quinone biosynthesis,phenylalanine metabolism,and phenylpropanoid biosynthesis.A total of 184 differentially expressed RNAs interacted at the binucleate phase.5.There were two mi RNAs were closely related to the two key genes obtained in the previous studies.In the binuclear stage,unconservative?chr3B?part2?17629 and unconservative?chr5b?part2?30016 have negative regulatory relations with Tae RPK and Tae Far5,respectively.Last,we predict fertility transformation mechanism,unconservative?chr3B?part2?17629 and unconservative?chr5B?part2?30016 inhibit the expression of Tae RPK and Tae Far5 respectively.When the mi RNA expression was blocked,m RNA can express to ensure the smooth progress of carbohydrate transport and metabolism,lipid transport and metabolism,and signal transduction,can ensure the demand for material and energy of the microspore development.In this way,normal fertility of microspores can be ensured,on the contrary,the up-regulated expression of mi RNA will inhibit m RNA expression,thus affecting anther development and thermosensitive fertility. |
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