Molecular Mapping Of Fertility Restoring Gene Rfk1-1B(CS)and Identification Of Genes Related To Fertility Restoration Of K-TCMS Wheat

Based on the two-line method,wheat KTM3315A is a K-type cytoplasmic temperature-sensitive male sterility?K-TCMS?wheat created by our group.It has the advantages of easy recovery,easy maintenance and no adverse cytoplasmic effect.It has important value in wheat breeding.Therefore,it is very important to breed its excellent,stable and highly recovering restorer line,which is of great significance in the study of wheat heterosis utilization.In order to solve the problem that K-TCMS wheat has high recovery and stable recovery system is still less and from the perspective of agricultural development,it is particularly important to study the molecular mechanism of K-TCMS fertility restoration gene and fertility restoration.In this study,on the one hand,the hybrid combination BC₁F₁ backcross population KTM3315A//China Spring/TM3315B constructed with K-type sterile wheat KTM3315A and Chinese spring as the parent was used as experimental material for fertility identification and genetic analysis.In addition,Molecular localization of the fertility restorer gene Rfk1-1B?CS?using molecular markers such as SSR,EST-STS and KASP aims to provide a theoretical basis for the genetic mechanism and breeding of K-TCMS wheat.On the other hand,the second-phase anthers of K-type wheat near-isogenic lines KTM3315A and KTM3315R,which were assembled by the research group for many years,were used as sequencing materials to analyze differentially expressed genes by bioinformatics,to identify important candidate genes and key metabolic pathways related to fertility restoration,and to provide useful information and new insights for studying the molecular regulation mechanisms of wheat fertility restoration.The main findings are as follows:1.The K-type wheat male sterile line KTM3315A,the restorer Chinese spring,KTM3315A/Chinese spring F₁ generation,and the near-isogenic line KTM3315R were used as the test materials.The anthers were observed by scanning electron microscopy and microspores I₂-KI staining analysis.The results showed that the anthers of KTM3315A were dry and thin,and the top did not crack and loose powder.The microstructure of the outer wall of the anther was disorderly arranged,and the microspores were deformed and wrinkled,showing a typical type of dyeing;The anthers of Chinese Spring,KTM3315A//Chinese Spring and KTM3315R were full and uniform,and the top end cracked loose powder.The outer wall of the anther was arranged neatly,the microspores were round and full,and the dyeing was uniform and the fertility was normal.It indicated that the fertility of K-type wheat male sterile line KTM3315A could be restored to normal in the presence of K-type wheat restorer gene Rfk1-1B?CS?.2.Genetic analysis and location analysis of fertility restorer genes in the BC₁F₁population of KTM3315A//China Spring/TM3315B.The results were as follows:The results of seed setting rate indicate that the fertility and sterile plants are close to 1:1,consistent with the fertility restoration controlled by a pair of major restorer genes?Rf?;The fertility restorer gene Rfk1-1B?CS?was mapped between KASP marker C28882165A and EST-STS marker BG274848,and their distance from the fertility restorer gene Rfk1-1B?CS?was 0.5 cM.One SSR molecular marker gwm413 was co-segregated with the fertility restorer gene Rfk1-1B?CS?.3.The transcriptome sequencing of the anthers of KTM3315A and its near-isogenic KTM3315R in the second nuclear phase was carried out,and a total of 2642 differentially expressed genes?DEGs?were obtained,of which 1404 were up-regulated and 1238 were down-regulated.Functional annotation analysis of these DEGs,GO significant enrichment analysis found that a large number of DEGs involved in metabolism,cell wall and enzyme activity.KEGG enrichment analysis showed that glucose metabolism,phenylpropanoid metabolism and biosynthetic pathways are closely related to fertility restoration.In addition,transcription factor analysis found that WRKY,bHLH and MYB transcription factors are closely related to the normal fertility of wheat,and they are involved in regulating the fertility restoration of wheat.4.The results of comprehensive analysis of functional annotations and key metabolic pathways showed that the sufficient content of sugar and flavonoids in KTM3315R anthers and the up-regulation of related enzymes in important metabolic pathways maintained the normal fertility of wheat compared with KTM3315A anthers.RT-PCR results consistent with transcriptome sequencing.