Screening And Preliminary Identification Of Genes Related To Grain Color And Fertility Of "Lanbiao" Male Sterile Wheat Material

The male sterile lines and AB lines of “Lanbiao” wheat were breeding selected by Chinese scholars such as Huang Shousong,by crossing the male sterile material found in the hybrid offspring of wheat varieties “72180” and “Xiaoyan 6” with the blue grain wheat selected by Li Zhensheng et al.The AB lines can be used for two purposes and solve the problem that the nuclear male sterility is difficult to maintain.The mechanism of fertility conversion and difference of seed color between the two materials of sterile lines and AB lines was explored in this study,using semi-thin sectioning technology,ultra-thin sectioning technology,high-throughput transcriptome sequencing technology and qRT-PCR.The conclusions were below:The results of phenotypic and cytological observation showed that there was no significant difference in morphology between male sterile lines and AB lines but in fertility by the time of flowering.The results of semi-thin section staining and transmission electron microscopy showed that the tapetal cells of sterile lines began to degrade in the early mononuclear stage,while the tapetal cells of AB lines remained intact.Finally,at the trinucleate stage,the development of microspores in the anther of the maintainer line was normal,but the mature microspores were not formed in the anther of the sterile line.It is speculated that the abortion of the sterile line may be caused by the early degradation of the tapetum,and the critical period of abortion may be mononuclear.The Statistical results showed that the RNA-Seq data had high quality.Enrichment analysis revealed that the differentially expressed genes in the two samples were highly enriched in phenylpropanoid biosynthesis,phenylalanine metabolism and nitrogen metabolism pathways,so these three biological metabolic processes may be related to the difference in fertility and blue grain traits of two samples.In the transcriptome sequencing data,a total of 71 differentially expressed genes related to anthocyanin were obtained through GO analysis and transcription factor family screening.Four structural genes in anthocyanin biosynthesis pathway were screened through KEGG analysis,and their expression values were significantly different between the two samples.From transcriptome sequencing data,36 differentially expressed genes related to fertility were obtained through GO analysis,and 9 differentially expressed genes related to fertility were further screened out by up-down regulated expression mode.Homology analysis revealed that three genes from the MYB,bHLH and WDR transcription factor families were highly homologous to the key regulatory genes for anthocyanin synthesis in corn or other plants.Fluorescence quantification showed that the expression levels of these three anthocyanidin-related genes in the maintainer samples were higher than those in the sterile lines,so they were predicted to be involved in the regulation of anthocyanin synthesis in blue kernels.The results of fluorescence quantitative analysis showed that among the nine fertility-related genes screened,the two genes with the annotations "MS1" and "CDC48A" showed significant differences in the expression levels of the two samples,so these two genes could be used as candidate genes for fertility recovery.The results of PCR identification showed that only the gene annotated as "MS1" in the two candidate genes existed in the genome of Thinpyrum ponticum,and the expression specificity and protein structure of MS1 gene in Thinpyrum ponticum(TpMS1)were highly similar to that of wheat MS1 gene,so it was speculated that TpMS1 gene might be the key gene for fertility recovery.The cloning of MS1 gene in the two samples showed that there were two MS1 gene sequences with different nucleotide sequences in maintainer line sample,one of which was consistent with the MS1 gene in sterile line sample and had a base difference on the first exon of the published wheat MS1 gene,and the other was consistent with the MS1 gene in Thinpyrum ponticum.Therefore,it was speculated that MS1 gene mutation was the cause of sterile line abortion.Further experimental verification is needed to prove the role of MS1 and TpMS1 genes in fertility transformation.