Fine Mapping And Molecular Clone Of Fertility Restorer Locus Rfk1 For Cytoplasmic Male Sterile Wheat KTP116A With Aegilops Kotschyi Cytoplasm

Wheat is listed as one of the crucial food crops in the world.Increasing wheat yield per unit area is of great significance to ensure the security of global food demand.With the per unit yield of wheat steadily growing,the heterosis was widely used for increasing wheat yield as one of the most effective ways,and cytoplasmic male sterility has become a vital mean of heterosis utilization in wheat.However,in the productive process of hybrid wheat seed,the traditional cytoplasmic male sterile materials meet various key problems,such as high cost of breeding and seed production,difficulty in guaranteeing purity and so on,which seriously hinder the research and development as well as utilization of hybrid wheat.To break through this bottleneck,KTP116A,a thermo-sensitive male sterile wheat with Aegilops kotschyi cytoplasm（TCMS-K）was created by our team,featuring obvious fertility conversion and the high critical temperature of transformation.It can adapt to the ecological conditions of wheat producing areas such as winter wheat region of Yellow River and Huaihe River valleys,Southwest winter wheat region,Northwest spring wheat region and Northeast spring wheat region in China,and it carries out the achievement of"one-linedual-uses",viz.functioningasboth male sterile line for F1 hybrid seed producing and inbreeding line for male sterile line repro ducing.It has great application potential and prospect in"two-line"hybrid wheat breeding.In the present study,aBC₁F₁ mapping population based on a cross derived from KTP116A and（LK783/TP116B）F₁ was constructed.The main restorer gene Rfk1 was fine mapped from wheat cv.LK783 by forward genetics and BSR-Seq strategy.By combining qRT-PCR and RNA-Seq analysis,the candidate genes of Rfk1 were predicted,cloned and identified,and the molecular markers closely linked to Rfk1 were selected for molecular marker-assisted selection breeding.The main results of this study are as follows:1.Scanning electron microscope observation,I₂-KI staining of anthers and pollen grains revealed that:In both the fertile BC₁F₁ individual plants and the restorer line LK783plants,the anthers were highly rounded with bifurcations at the top,and the outer surface was glossy with fibers arranged neatly.The Ubisch bodies were distributed equably with abundant sporopollenin.And the pollen grains were stained dark blue–black.However,the anthers from KTP116A were shrunken and not cracked with disrupted.In addition,the arrangement of fibrous cells in the exine was disordered.The Ubisch bodies in the inner wall is scattered,and the edge of the pollen grain can not be fully stained by I₂-KI.2.Through SNP-index method and linkage analysis,Rfk1 was delimited in a 6.9 cM interval between PCR marker Xnwafu＿6 and SSR marker Xbarc137.Furthermore,KASP markers were designed based on the SNPs in preliminary mapping interval.By combining other molecular markers,The Rfk1 gene was finally located within a region of 26.0 Mb between Xnwafu＿6 and Xnwafu＿1B32.Next,the differentially expressed genes in anthers in this region were screened by wheat eFP database and qRT-PCR,and Traes CS1B02G197400LC was determined to be the most likely candidate gene.3.The sequence analysis results showed that Traes CS1B02G197400LC measured1,350 bp in length with only one exon.The predicted protein contains a conserved PMEI＿like domain and it belongs to the pectinesterase superfamily.Alignment of the Traes CS1B02G197400LC gene sequence in LK783 and KTP116A detected 10 single non-synonymous SNPs in the exon.In addition,a 7 bp deletion was detected at 1,177 bp in KTP116A,which may have been caused a change in the structure of the encoded protein.Expression pattern further revealed that Traes CS1B02G197400LC was expressed in root,stem,leaf,and anther in KTR116A and LK783,it was only differentially expressed in anther.Compared with KTP116A,the Traes CS1B02G197400LC expression levels in LK783 were significantly upregulated during trinucleate stage.4.By screening the molecular markers in the candidate interval,a molecular marker Xnwafu＿4 which is closely linked with Rfk1 was obtained.The marker showed stable polymorphism between restorer lines and male sterile lines.According to self-setting rate data and PCR analysis results for the marker Xnwafu＿4,the accuracy of the marker was90.4%in 2018 and 94.8%in 2019.