Establishment Of Abortion Regulatory Network And Function Analysis Of TaUGP1-6A In Isonuclear Alloplasmic Male Sterility Wheat (Triticum Aestivum L.)

At present,hybrid wheat breeding is a very promising strategy to maximize the use of heterosis and improve wheat yield.Cytoplasmic male sterility?CMS?,as the main way to utilize wheat heterosis,could not produce functional pollen in crops,thus providing ideal materials for the study of cytoplasmic inheritance,reproductive growth and pollen development.In recent years,a set of ideal experimental materials of isonuclear heterogenic male sterile wheat?IAMSLs?have been studied and found they have the advantages of stable sterility,no haploid production,improving wheat quality,enhancing powdery mildew resistance and increasing growth potential.Thus,they are excellent economic male sterile lines and have great application potential in hybrid breeding of wheat.However,the mechanism of pollen abortion is still unclear in these economical IAMSLs wheat,especially the core molecular mechanism of pollen abortion.In view of this,the 1BL/1RS karyotypes of five IAMSLs?K706A,Va706A,Ju706A,C706A and U706A?and their maintainer B706 were identified in this study.Phenotypic and cytological observations of plants,anthers,tapetum,microspore and pollen wall were carried out by using microscopic and submicroscopic techniques at different developmental stages of IAMSLs and maintainer.The dynamic changes of programmed cell death?PCD?were detected by TUNEL and DNA ladder techniques in tapetum.The transcriptome sequencing analysis was conducted in anthers at the abortive critical stage,and the results were further verified by physiological index determination and quantitative analysis.The candidate gene TaUGP1-6A-6A was obtained by transcriptome analysis and the full length of this gene was further amplified by RACE.The transform of Arabidopsis thaliana and VIGs silencing technology was analyzed to define the function of TaUGP1-6A.Main contributions of this work are as follows:1.Molecular marker electrophoresis results showed that all the five IAMSLs were all1BL/1RS translocation lines.Cytological studies of the five IAMSLs and their maintainer showed that the pistils of the male sterile lines developed normally and had the ability to accept pollen during the whole growth and development process,however,the anther of IAMSLs showed some abortive traits,including obvious dryness,no pollination,and internal pollination during the trinucleate stage.I₂-KI staining showed the abortion types were typical abortion and stainable abortion,but the proportion is different in IAMSLs.After the late uninucleate stage,the microspore began to develop disorderly in IAMSLs,but the reasons for abortion are different.Tissue sections,TUNEL and DNA ladder were carried out to observe tapetum degradation and detect PCD,and the results indicate that the tapetal cell PCD was advanced in K706A and others were delayed.Ultrastructural observation showed that sporopollenin could not be synthesized due to the absence of tapetum elaioplast in late uninucleate stage.These results indicate that the abnormal PCD of tapetum and the absence of elaioplast are the main causes of pollen abortion.2.High quality clean data of 109.43 GB were obtained by transcriptome sequencing analysis of IAMSLs and maintainer at the binuclear stage.Contrasted with B706,4,294,15,835,5288,20,444,10,136 differentially expressed genes?DEGs?were obtained from K706A,Va706A,Ju706A,C706A and U706A,respectively.A core gene set containing1,392 DEGs was found by Venn diagram,which included 172 up-regulated genes and 1,220down-regulated genes in each IAMSLs.Through KOG,GO,KEGG and WGCNA analysis,important pathways related to abortion?glycometabolism,oxidative phosphorylation,phenylpropane biosynthesis,phosphatidylinositol signal?and important candidate genes?GLTP,Pectinesterase,UGPase?were obtained.The results of physiological indices,histochemical analysis and quantitative analysis showed that the decreased sugar and energy contents,the excessive accumulation of ROS,the block of sporopollenin synthesis and the abnormal PCD of tapetum were appeared due to the influence of down-regulated genes in these pathways.Based on the above results,a core transcription interaction network was constructed to regulate pollen abortion in IAMSLs wheat.3.Two copies of TaUGP1gene,TaUGP1-A and TaUGP1-B,were cloned using the maintainer anther cDNA as template.Sequence alignment revealed that there were 17 SNPs between TaUGP1-A and TaUGP1-B cDNA sequence and the encoded amino acids remained basically unchanged.Based on transcriptome analysis,the DEG?Traes₆AS₃A8E07254?encoding UGPase was located on chromosome 6A.Therefore,a 1,731 bp full-length DNA sequence of TaUGP1-6A was cloned from B706 by RACE.Sequence analysis showed that TaUGP1-6A encoded 467 amino acids and had a typical UDPGP conservative domain.To verify the TaUGP1-6A function,we constructed an overexpression vector pCAMBIA3301-TaUGP1-6A and transformed the homozygous mutant of Arabidopsis thaliana?Atugp1atugp1/Atugp2atugp2?.The results showed that sterile plants?atugp1atugp1/atugp2atugp2?appeared in the offspring of the untransformed heterozygotes,while the progenies of the transformed plants were all fertile.Furthermore,the results indicated that the complementation of the gene compensated for the deletion of AtUGP1 gene in Arabidopsis thaliana and confirmed that TaUGP1-6A was related to fertility.The TaUGP1-6A of B706plants was silenced effectively by BSMV-VIGS technology,and the sterile characters of anther non-splitting appeared in the silent plants,and the self-fertility rate was also decreased.These results indicated that the expression of TaUGP1-6A was closely related to the fertility of IAMSLs in wheat.