Fine Mapping And Candidate Gene Identification Of Male Sterility Mutant Gene BrABCG26 In Chinese Cabbage(Brassica Rapa L. Ssp. Pekinensis)

Male sterility refers to the phenomenon that the stamens can not produce normal active pollen,but the pistils can normally develop.Using male sterility lines to prepare hybrids is an important way to utilize the heterosis of Chinese cabbage（Brassica rapa L.ssp.pekinensis）.In this study,0.8%EMS solution was used to mutagenize the germinated seeds of a Chinese cabbage DH line‘FT’,and a stably genetic male sterility mutant was obtained,which was named as msm3（male sterility mutant 3）.Based on the morphological identification and genetic analysis of mutant msm3,the candidate genes were identified by Mut Map mapping method combined with KASP genotyping technology,and the clone and expression characteristics of candidate genes were analyzed.The main results are as follows:1.At the vegetative growth stage,there was no significant difference between the wild-type‘FT’and the mutant msm3.At the reproductive growth stage,the floral organs of the wild-type‘FT’and the mutant msm3 were compared and observed,indicating that the anther of the mutant msm3 was shrivelled and had no pollen,and the flower bud,sepal,petal and stigma of the mutant msm3 were also smaller than those of the wild-type‘FT’;Pollen viability detection revealed that there was no pollen grains in mutant msm3;Paraffin section results showed that the failure of vacuolation of the mononuclear microspore,accompanied by abnormal tapetal degradation,resulting in the anther abortion of mutant msm3.2.F₁generations were obtained by crossing the mutant msm3（female parent）and wild-type‘FT’（male parent）.F₁generations were fertile plants,and F₂generations were obtained by self-crossing F₁generations.In F₂generation,the ratio of fertile plants to sterile plants was 3.08:1,indicating that the mutant msm3 was controlled by a pair of recessive nuclear genes.3.The high-throughout sequencing was performed on two parents（wild-type‘FT’and the mutant msm3）and 50 male sterility plants of F₂generation.The mutant gene was located on chromosome A01 by Mut Map method,and the length of candidate region was 3.84 Mb（A01:23,573,818-27,410,787）,which contained 4candidate SNPs.Bra A01g038270.3C was further identified as the candidate gene by KASP genotyping technology,Bra A01g038270.3C was homologous to AT3G13220 in Arabidopsis and encoded transporter ABCG26,which plays an important role in pollen wall formation,and named as Br ABCG26 in this study.4.The full length and coding sequence of the candidate gene Br ABCG26 were cloned and sequenced in wild-type‘FT’and mutant msm3,the results showed that a C→T mutation occurred on the second exon of the candidate gene Br ABCG26,resulting in the premature termination of amino acid coding.5.Quantitative real-time PCR analysis showed that candidate gene Br ABCG26was expressed in all organs of wild-type‘FT’and mutant msm3,with the highest expression in flower buds.Compared to the wild-type‘FT’,the expression of Br ABCG26 gene in flower buds and anthers was significantly reduced in mutant msm3;Promoter activity analysis showed that Br ABCG26 gene was expressed in roots,stems,leaves and flower buds,a strong GUS signal was detected in flower buds;Subcellular localization indicated that Br ABCG26 was a nuclear localization protein.6.The expression levels of genes involved in pollen wall development（AMS,ACOS5,MS2,CYP704B1 and CYP703A2）were different between wild-type‘FT’and mutant msm3.Compared with wild-type‘FT’,the expression of MS2,CYP704B1 and CYP703A2 in mutant msm3 was significantly reduced.