Multi-Omics Analysis Of The Mechanism Of Pollen Abortion In 'Hua Nong' Seedless Ponkan

Citrus production and planting area are the largest among various fruit trees,mainly used for fresh food or juice.Seedless citrus has greater market competitiveness due to its convenient consumption and simple processing.The main reasons for citrus seedlessness are male sterility,embryo sac abortion,embryo abortion or self-incompatibility,and the most common reason for male sterility is pollen abortion.There are many ways to regulate pollen abortion in herbaceous plants such as Arabidopsis and rice,but the potential molecular regulation mechanism of pollen abortion in most woody plants such as citrus is still unknown.In this study,'Hua Nong' seeded ponkan(WT)and its natural seedless mutant(MT)were used as materials to analyze the reasons for the formation of'Hua Nong' seedless ponkan and its molecular regulation mechanism of seedleesness by morphological and multi-omics analysis.The main results are as follows :1.Morphological analysis of anthers development process found that the anthers powder of 'Hua Nong' seedless ponkan is less than that of the wild type,and most of the pollen grains are inactive,unable to germinate in vitro and in vivo.During pollen development,the meiosis process was abnormal.Chromosomes in metaphase I of meiosis could not be arranged in the equatorial plate,and segregation distortion occurred in late stage of meiosis I.There were 2-7 micronuclei in callose at tetrad stage.The cytoplasm of pollen grains began to degrade in early uninucleate stage,and the nucleus disappeared in late uninucleate stage.To the mature stage,the pollen grains size is not uniform with obvious differences in size,and the groove is not clear.The shape is not full enough,some even collapse.The mature pollen grains contain some or no inclusions,and eventually formed abortive pollen grains.2.Transcriptome sequencing(RNA-Seq)was performed on 18 samples of 'Hua Nong' seedless ponkan and 'Hua Nong' seeded ponkan at premeiotic(Stage 1),meiosis(Stage 2)and maturation stages(Stage 3),and 186.24 Gb Clean Data was obtained.There were 5,421 differentially expressed genes(DEGs)in the three periods,and the number of DMGs in the mature stage is the largest.The number of down-regulated DEGs were more than up-regulated genes in the three periods.Compared with 'Hua Nong' seeded Ponkan,a total of 2,025 DEGs were up-regulated and 3,794 DEGs were down-regulated in 'Hua Nong' seedless Ponkan during the three periods,with 98 common differential genes.Many DEGs are ERF,NAC,WRKY transcription factors.The enrichment analysis of metabolic pathways showed that many DEGs were related to metabolic pathways such as starch,sugar and hormones.Quantitative analysis showed that starch and soluble sugar contents in anthers of 'Hua Nong' seedless ponkan were significantly lower than those of'Hua Nong' seeded ponkan at maturity.Hormones determination showed that IAA,JA and ABA contents were also significantly lower.Those indicated that the decrease of starch,sugar and hormone may be related to pollen abortion of 'Hua Nong' seedless ponkan.3.Whole-genome bisulfite sequencing(WGBS)was performed on 12 samples in three critical stages of anthers development of 'Hua Nong' seedless ponkan and its wild-type.On average,11.26 Gb clean Date were was obtained for each sample,and the total amount of data was 135.12 Gb clean Date.There are 50,845 differentially methylated regions(DMRs)in the three periods,corresponding to 15,426 differentially methylated genes(DMGs).The methylation level of CHH was the highest among CG,CHG and CHH sequences context.The methylation level of 'Hua Nong' seedless Ponkan was higher than its wild-type in the three periods,especially in the pre-meiosis stage.In different regions of the genes,methylation occurs mainly in the promoter region.There were 1106 transcription factors in DMGs in the three periods,among which MYB,ERF,b HLH were the most common.GO enrichment analysis showed that DMGs were mainly related to membrane components and membrane proteins.KEGG enrichment analysis found that many DMGs were enriched in starch,sugar and hormone pathways,similar to DEGs.In addition,the methylation inhibitor Zebularine(Zeb)treatment could partially restore the pollen fertility of 'Hua Nong' seedless Ponkan.These results suggest that methylation may be involved in regulating pollen development of 'Hua Nong' seedless ponkan.4.Metabolomics analysis of 'Hua Nong' seedless ponkan and its wild-type in three critical periods of anthers development.A total of 10 categories of 639 metabolites and197 differential metabolites(DMs)were detected during the three periods,with the largest proportion of flavonoids.There are only 6 common differential metabolites,and72,50 and 131 differential metabolites in the three periods,respectively.During the Stage1 and Stage 2,the number of up-regulated metabolites was slightly more than the number of down-regulated.However,the number of DMs in the mature stage is the largest,and the number of down-regulated metabolites was significantly more than that of up-regulated metabolites.KEGG enrichment analysis showed that the different metabolites in the three periods had a high degree of enrichment in the metabolic pathway of secondary metabolite.Correlation analysis showed that flavonoids dominated the correlation between metabolites.In addition,pollen fertility of 'Hua Nong' seedless ponkan decreased after flavonoids treatment.These results indicate that flavonoids may also play an important role in pollen development,but it may has different functions in different periods.5.Transcriptome,methylome and metabolomics Transcriptome were performed at three critical stages of anthers development.Most of the differentially expressed genes in the transcriptome were methylated,and the gene expression levels were negatively correlated with the degree of methylation.KEGG enrichment analysis of the shared differentially genes DEG/DMGs showed that these genes were mainly involved in MAPK singaling,plant hormone,starch,sugar and cell cycle pathways.In the pre-meiosis stage of 'Hua Nong' seedless ponkan anthers,many genes related to meiosis,hormone and flavonoid biosynthesis were methylated or demethylated,and metabolomics also found that most of the differential metabolites are flavonoids.These results suggest that methylation may affect pollen fertility of 'Hua Nong' seedless ponkan by regulating the expression of some related genes.