Study On The Fertility Regulation Mechanism Of A Multiple Allele Genetic Male Sterile Line In Chinese Cabbage

Chinese cabbage is a bisexual cross pollinated crop with significant heterosis.The utilization of male sterile lines is a good way of hybrid seed production.In 1992,our team found a Chinese cabbage genic male sterile material with multiple allele inheritance.The male sterile lines with 100%sterile plant rate were screened by test crossing,and the“multiple allele genetic hypothesis of nuclear male sterility in Chinese cabbage”was put forward,and realized the directional transfer of a variety of Chinese cabbage vegetables such as milk cabbage,purple tsai-tai,savoy and flowering Chinese cabbage.In the early stage of this study,several molecular markers linked to Ms gene were developed to locate Ms gene on A07chromosome.By screening,sequencing and splicing the BAC library of male sterile plants（Ms Ms）in Chinese cabbage,and comparing it with the reference genome,it is found that there are large fragments of inversion and insertion in the location region of male sterile gene,indicating that there may be complex structural variation at this fertility site.In order to reveal the molecular mechanism of male sterility inherited by multiple alleles in Chinese cabbage,this study used the male sterile plant（Ms Ms）of the genetically stable male sterile dual-purpose line and restorer line（Ms^fMs^f）as parents to construct F₂isolated population.The Ms gene was fine located by developing molecular markers and combined with the whole genome resequencing of the parents,and the function of the candidate gene was verified and the temporal and spatial expression pattern was analyzed.The restorer line（Ms^fMs^f）,male sterile plant of dual-purpose line（Ms Ms）and maintainer line（msms）were resequenced in the second and third generations.The main results are as follows:1.Morphological observation of flower organs of restorer line‘AB04-F’and male sterile plant of dual-purpose line‘AB03-S’showed that the flower organs of‘AB03-S’were small,the stamens were shriveled,and there was no pollen release.The pollen mother cells of their anthers gathered at the tetrad stage,the tapetum expanded continuously,squeezed the microspores,and finally the anthers abortion.However,there were a large number of pollen on the surface of‘AB04-F’anthers,indicating that their anthers could normally undergo meiosis and released energetic pollen grains.2.Using‘AB04-F’and‘AB03-S’to construct the F₂generation isolation population,the Ms gene was located in the 231.3 kb region of A07 chromosome,including 21 genes.Combined with the SNP and In Del analysis of parental whole genome resequencing,it was found that only Bra A07g008890.3C had an In Del in the exon,which encodes the FHA domain containing protein.Its Arabidopsis homologous gene AT1G34355 is related to pollen development.It is predicted that Bra A07g008890.3C is the candidate gene of Ms.3.In‘AB03-S’,the third exon of Bra A07g008890.3C has a 9 bp base sequence insertion,which contains a termination codon,resulting in early termination of translation.Linkage analysis showed that the In Del was co separated from the male sterile trait.Through the heterologous transformation function complementarity experiment of Arabidopsis,it was proved that the fertile parent of Chinese cabbage Bra A07g008890.3C gene can restore the stamen fertility of Arabidopsis homologous gene mutant,so as to verify its function.4.Promoter activity analysis showed that Bra A07g008890.3C was mainly expressed in flower organs;q RT-PCR analysis showed that the expression of Bra A07g008890.3C in fertile buds was significantly higher than that in sterile buds,and it was highly expressed in stamens;Ms^fMs^fand Ms Ms anther transcriptome analysis showed that the expression of Bra A07g008890.3C in Ms^fMs^fanthers was significantly higher than that in Ms Ms.Bra A07g008890.3C promoter region contained MYB and WRKY transcription factor cis-acting elements,and 12 specifically expressed WRKY and 18 specifically expressed MYB transcription factors were identified in Ms Ms.Most of the differentially expressed genes between Ms^fMs^fand Ms Ms anthers were significantly enriched in plant-pathogen interaction,phenylpropanoid biosynthesis,MAPK signaling pathway-plant,starch and sucrose metabolism,and pentose and glucuronate interconversions metabolic pathways.5.The second and third generation resequencing analysis of Ms^f,Ms and ms homozygous materials of Chinese cabbage and milk cabbage showed that within the multiple allele mapping interval,there were less SNPs and In Dels between restorer lines and male sterile lines,but more SNPs and In Dels between them and maintainer lines.Some non synonymous SNPs and frameshift mutant In Dels caused gene coding changes.There are 35bp-7,520 bp fragment insertions and deletions between different genotypes of Chinese cabbage and milk cabbage,and there are large fragment inversion and fragment duplication between different genotypes and different genetic background materials,reflecting the sequence complexity of the regulatory region of multiple alleles,which may be an important reason why the fertility restoring gene Ms^fand sterility maintaining gene ms in the multiple allele series have not been cloned so far.