Identification Of A Genetic Male Sterile Mutant Ftms In Chinese Cabbage (Brassica Campestris Ssp. Pekinensis)

The phenomenon of male sterility is widely in higer plants.Plant male sterility is one of the important methods of hybrid seed production for cruciferous plant with significant hybrid vigor.Scientists have long been interested in research on plant male sterility.As spontaneous male sterile mutant is relatively rare,artificial mutagenesis techniques have become an effective method to create male stertile material.Chinese cabbage（Brassica campestris ssp.pekinensis）is an important vegetable of Cruciferae with remarkable heterosis.In our study,a doubled haploid（DH）line of Chinese cabbage variety was employed as experimental material to create a male sterile mutant ftms by ⁶⁰Coγ-ray mutagenesis combined with the isolated microspore cultures.Map-cloning strategy was employed to identify the mutant gene ftms by using high throughput sequencing as a reference.The main research results are as follows:1.Phenotypic and genetic characteristics of male sterile mutant ftms in ChinesecabbageFor genetic analysis,the mutant ftms was used as a female parent crossed with its wild type‘FT’to produce F₁ and F₂ progenies.The results indicated that the male sterile phenotype of the mutant ftms was controlled by a recessive nuclear gene.At the vegetative stage,the mutant ftms was no obvious differences with the wild-type‘FT’.In reproductive stage,anther development of mutant ftms was degraded and no mature pollen grain was released.Using paraffin section observation,the abortion of mutant ftms began at the terad stage.The tapetum cells characterized as abnormally expanded and highly vacuolated resulted in the microspore degradation.2.Mapping of mutant gene ftms by BSR-SeqThe mutant ftms with white petal and Stalk Chinese cabbage DH line‘14S701’with yellow petal were used as the parents to construct F₂ mapping population.There were four floral organ phenotypes in F₂ population,including male fertility with yellow petal,male sterility with yellow petal,male fertility with white petal and male sterility with white petal.For genetic analysis,two traits,male fertility and petal color were controlled by a recessive nuclear gene,respectively and characterized as inherited independently.Two RNA pools were constructed,including male fertility with yellow petal pool（YF-pool）and male sterility with white petal pool（WS-pool）.Each pool consisted of fifty individuals,respectively.By BSR-Seq analysis,two genes,male fertility and petal color were mapped simultaneously.The candidate regions linked to the target traits were obtained on A05 and A02 chromsomes,repectively.By SSR markers development in candidate regions,the mutant gene ftms were mapped to A05 chromsome,and the white petal gene mapped to the A02 chromsome.3.Fine mapping of mutant gene ftms by large-scale segregation linkage analysisA large F₂ popualtion with 2,600 homozygous male sterile individuals were developed to further narrow the mapping region.The mutant gene ftms was finally mapped between InDel20 and InDel14 markers on A05 chromsome at agenetic distance of 0.02cM and 0.06cM,respectively.Compared with Brassica rapa genome,114 genes were predicted in a physical distance of approximately 1.7Mb invterval.Due to the canadidate region is located near the centromeres of A05 chromsome,the recombination rate is extremely low.It has resulted in the failure to use molecular marker linkage analysis to further narrow the canadidate region.4.Candidate gene prediction based on whole genome resequencingWhole genome resequencing was performed on mutant ftms and wild-type‘FT’.Using the sequencing information,the mutation loci on mutant ftms were detected.In the candidate region,a total of 82 homozygous SNP and InDel mutation locus were screened,of which only one non-synonymous SNP were dected in the exon region.This gene,Bra010198 encodesβ-（1,3）-galactosyltransferase,which is in involved in the pollen exine development.In mutant ftms,a G→T base transversion in the fifth exon of Bra010198 resulted in the 281^th amino acid Gly（GGG）mutating Val（GTG）whthin the conserved domain.q RT-PCR analysis showed that the candidate gene Bra010198 was specifically expressed in stamens.5.Family members identification of the canadidate gene Bra010198Using the protein sequences of 20β-（1,3）-galactosyltransferase family genes identified in Arabidopsis thaliana as reference,a total of 30β-（1,3）-galactosyltransferases were identified in Brassica rapa.We found that theβ-（1,3）-galactosyltransferase family gene in Brassica rapa have a good collinearity relationship with homologous gene in Arabidopsis thaliana.Phylogenetic analysis showed that theβ-（1,3）-galactosyltransferase family genes in Brassica rapa were divided into two groups:group I and group II.Physicochemical properties,conserved motifs and exon/intron numbers of the family member were relatively consistent in each group.6.Transcriptome analysis of male sterile mutant ftms by RNA-SeqUsning RNA-Seq technology,compatative transcriptome analysis was performed on the buds of mutant ftms and wild-type‘FT’.A total of 2,305 differential expressed genes were detected.These genes were all up-regulated,including tapetum development related gene AMS,callose synthesis related genes CALS5 and CDKG1,primexine development related gene RPG1,and pollen wall formation related gene families,including 3 AGPs,13 PLs,25 PMEs,7 PMEIs and 18 PGs.We performed GO and KEGG functional enrichment analysis.GO terms associated with pollen and poolen tube development,cell and pollon wall,carbohydrate metabolism and lipid metabolim were significantly enriched;and multiple carbohydrate metabolism and lipid metabolim related pathways were aslo significantly enriched.Therefore,the mutation of Bra010198 encodedβ-（1,3）-galactosyltransferase might be affect the synthesis of AGPs,which related to the abnormal development of tapetum,callose,primexine and pectin metabolic,resulting in stamen abortion of male sterile mutant ftms in Chinese cabbage.