Analysis Of Ovary DNA Methylation During Delayed Fertilization In Hazel Using The Methylation-sensitive Amplification Technique

Hazelnuts(Corylus avellana L.)are edible nuts that have great economic value and unique delayed fertilization characteristics.The early ovary of hazel starts to develop only after pollination,and a growth signal from pollen tubes is thought to be the key factor that triggers this process.DNA methylation plays an important role in the epigenetic regulation of gene expression in eukaryotes,and although DNA methylation may play an important role in activating ovary development,changes in DNA methylation during the delayed fertilization process in hazel remain unknown.In this study,the DNA methylation changes during pre-and post-pollination stages were assessed using a methylation-sensitive amplification polymorphism(MSAP)assay.In total,760 CCGG amplified sites were detected by MSAP assay.The total methylation ratio of hazel was relatively high,varying from 44.61% to48.68%.After pollination,the total methylation ratio decreased slightly by approximately 4%.There were significant differences in MSAP bands at pre-and post-pollination stages.After MSAP analysis,15 specific differentially displayed DNA fragments were cloned for sequence analysis.Most of the genes were homologous to mitochondrial complete sequence or associated with ovary development.The expression levels of fragments L8 and L10 were closely related to the demethylation and methylation changes.When the demethylation and methylation processes were activated,the expression levels were significantly increased and decreased,respectively.In particular,frequent DNA methylation and demethylation changes of the same fragment were detected during different development stages,which led to the inactivation or activation of specific genes.In conclusion,the epigenetic changes in the genome probably represent an important mechanism that initiates development of the ovary and ovule after pollination.