| As an important way to increase yield in wheat,heterosis plays an important role in wheat breeding practice.Cytoplasmic male sterility(CMS)is an important means to utilize heterosis,and it is of great significance to promote the research and utilization of heterosis in wheat.Ju706A is a D~2 cytoplasmic male sterile line,which has the advantages of strong recoverability and disease resistance.It has a wide range of application prospects in breeding practice,but its fertility regulation mechanism is not clear.Therefore,in this study,the sterile line Ju706A,its isotype maintainer line 706B and restorer line LK783 were used as test materials to observe the phenotypes of florets,anthers,and microspores;to construct a backcross population Ju706A//LK783/706B,preliminary localization of the fertility restorer gene Rf using BSA and SSR molecular markers;to use wheat 660K gene chip typing technology and the developed SNP molecular markers to narrow down the candidate region,finely map Rf,and use the transcriptome Data and real-time fluorescence quantification(q RT-PCR)to screen and identify candidate genes;at the same time,based on the cloned sequence differences,design specific In Del markers for molecular marker-assisted selection;Through subcellular localization of candidate genes and the use of barley stripe mosaic virus-induced gene silencing(BSMV-VIGS)technology for preliminary functional verification of candidate genes,the following research results are obtained:1.The phenotype of the sterile line Ju706A,the restorer line LK783 and its F1generation were observed,the results showed that the stamens and pistils of LK783 were developed normally,and the pollen grains were completely stained by I2-KI solution;the anthers of Ju706A were thin and incompletely stained by I2-KI solution,showing the characteristics of typical abortion and stainable abortion;F1 showed normal pistil and stamen,and most pollen grains can be stained by I2-KI.Scanning electron microscope observation of anthers(trinucleate stage)of Ju706A and LK783 showed that LK783 had normal outer epidermis,inner epidermis and microspores;while Ju706A anthers had irregularly arranged outer epidermis,sparse ubisch bodies in inner epidermis,small and shrunken microspores.The above showed that the male fertility of Ju706A can be restored by LK783,and its F1shows similar phenotypic characteristics to LK783.2.The sterile plants and fertile plants of Ju706A//LK783/706B backcrossed progeny population showed a 1:1.2 segregation ratio.According to the Chi-square test,the fertility restoration of Ju-type cytoplasmic male sterile wheat conforms to Mendel’s separation of a pair of genes,and its fertility restoration is controlled by a pair of dominant genes.Using 356pairs of SSR primers to screen for polymorphic markers in parents and DNA mixed pools,a total of nine pairs of SSR primers were linked to the restorer gene Rf,and they were all located on 1B chromosome.The nine pairs of SSR primers were tested in sterile plants and a genetic linkage map was constructed.The results showed that the restorer gene Rf was closely linked with Xgwm18 and Xgpw1143 markers,and the genetic distances were 4.1 c M and 10.7 c M,respectively.3.The typing results of wheat 660K gene chip showed that 27%of the differential SNP loci were concentrated on 1B chromosome,which was consistent with the preliminary mapping results.By designing specific SNP markers and screening in parents and DNA mixed pools,six polymorphic markers were found to be closely linked to the target gene,namely AX-94768879,AX-95117169,AX-174254104,AX-111201011,AX-94793363,AX-111281507.According to the polymorphism markers and the number of recombinant individuals,the restorer gene Rf was narrowed to the 2 Mb interval between SNP markers AX-174254104 and AX-111201011.This interval contained 19 candidate genes.Through RNA-Seq data and gene expression levels in each tissue,the candidate genes were locked to Traes CS1B02G197400LC.The gene(temporarily named TaRfd1)encoded a pectinesterase/pectinesterase inhibitor,which was highly expressed in anthers and had the highest expression level in the trinucleate stage of fertile pollen.4.Using the anther c DNA of LK783 and Ju706A as templates,TaRfd1 was successfully cloned.Sequence analysis showed that there were 23 SNP differences and 7 bp deletion(CCGGGGG)between LK783 and Ju706A.This 7 bp deletion resulted in a change in the amino acid sequence starting from the 393th amino acid.Based on the 7bp deletion in Ju706A,an Indel marker Xnwafu1 was designed,which can identify 100%of all sterile individuals in the BC1F1 population,97.6%of maintainer lines,93.4%of restorer lines,and70.3%of test materials.The phylogenetic tree analysis showed that the gene was closely related to the homologous gene of Aegilops tauschii subsp.tauschii and may have similar functions.5.The results of subcellular localization showed that pectin methylesterase was localized on the cell wall.Effectively silence the TaRfd1 gene through barley stripe mosaic virus infection(BSMV-VIGS).Phenotypic observation showed that after the TaRfd1 gene was silenced,the florets were thin,the anthers could not crack,the I2-KI staining was incomplete,the sperm nucleus were round,the pollen grains were shrunk,and the pollen germination rate decreases;cytological observations showed that after the TaRfd1 gene was silenced,outer epidermis was irregularly arranged,sparse ubisch bodies,shrunken microspores,delayed degradation of tapetum cells of anthers,irregular arrangement of the outer wall of microspores,abnormal sporopollenin deposition;gene expression,seed setting rate survey and pectin content determination results indicate d that in silent plants,the expression of TaRfd1gene decreased,the seed setting rate decreased,and the pectin content increased.The above results indicated that the TaRfd1 gene was closely related to fertility. |
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